Oligonucleotide compounds for targeting huntingtin mrna

ABSTRACT

This disclosure relates to novel huntingtin targets. Novel oligonucleotides for the treatment of Huntington&#39;s disease are also provided.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/811,580, filed Mar. 6, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/263,200, filed Jan. 31, 2019, now U.S. Pat. No.10,774,327, which is a continuation of U.S. patent application Ser. No.15/697,120, filed Sep. 6, 2017, now U.S. Pat. No. 10,435,688, which is acontinuation of U.S. patent application Ser. No. 15/089,319, filed Apr.1, 2016, now U.S. Pat. No. 9,809,817, which claims priority to U.S.Provisional Patent Application Ser. No. 62/289,274, filed Jan. 31, 2016,and U.S. Provisional Patent Application Ser. No. 62/142,731, filed Apr.3, 2015. The entire contents of these applications are hereinincorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numbersNS038194 and TR000888 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 29, 2021, isnamed 724653_UM9-203CON4_ST25.txt and is 311,021 bytes in size.

FIELD OF THE INVENTION

This disclosure relates to novel huntingtin targets and noveloligonucleotides for the treatment of Huntington's disease.

BACKGROUND

Neurological disorders including Huntington's disease, Parkinson'sdisease and Alzheimer's disease represent a major unmet medical need. Insome cases, these diseases are monogenic, making them ideal targets foroligonucleotide therapeutic intervention, e.g., RNA interference (RNAi).RNAi is a fundamental mechanism involving short double stranded RNAfragments that can be used to reprogram cellular machinery and silenceand degrade targeted mRNA on demand. This technology is clinicallyadvanced and has revolutionized the field of human functional genetics.

Many different technologies have been explored for mRNA knockdown bothas therapeutics and as tools for functional study, including viral baseddelivery of short hairpin RNAs (shRNAs), antisense oligonucleotides(ASOs), and naked or slightly modified siRNAs (Sah, D. W. Y. & Aronin,N. Oligonucleotide therapeutic approaches for Huntington disease. J.Clin. Invest. 121, 500-507 (2011); DiFiglia, M. et al. Therapeuticsilencing of mutant huntingtin with siRNA attenuates striatal andcortical neuropathology and behavioral deficits. Proceedings of theNational Academy of Sciences of the United States of America 104,17204-17209 (2007)).

ASOs have also shown to be a promising approach. This technologyexhibits efficient delivery to cells without a delivery vehicle and hasbeen administered to brain for the treatment of Huntington's disease forsuccessful knockdown in both rodent and non-human primate brains(Mantha, N., Das, S. K. & Das, N. G. RNAi-based therapies forHuntington's disease: delivery challenges and opportunities. Therapeuticdelivery 3, 1061-1076 (2012); Kordasiewicz, H. B. et al. SustainedTherapeutic Reversal of Huntington's Disease by Transient Repression ofHuntingtin Synthesis. NEURON 74, 1031-1044 (2012)). Unfortunately,current studies show that a 700 μg cumulative dose administrated overtwo weeks is required to see just 50% silencing (Kordasiewicz, Supra).

Unmodified siRNA (“naked siRNA”) has been difficult to deliver to moresensitive cell lines and in vivo to tissue in the past. Althoughtransfection reagents such as Lipofectamine can be used, there is a verynarrow window within which it is efficacious and non-toxic, and it mustbe optimized independently for different batches of neurons to determinesiRNA to lipid ratios necessary for comparable levels of silencing(Bell, H., Kimber, W. L., Li, M. & Whittle, I. R. Liposomal transfectionefficiency and toxicity on glioma cell lines: in vitro and in vivostudies. NeuroReport 9, 793-798 (1998); Dass, C. R. Cytotoxicity issuespertinent to lipoplex-mediated gene therapy in-vivo. Journal of Pharmacyand Pharmacology 1-9 (2010); Masotti, A. et al. Comparison of differentcommercially available cationic liposome-DNA lipoplexes: Parametersinfluencing toxicity and transfection efficiency. Colloids and SurfacesB: Biointerfaces 68, 136-144 (2009); Zou, L. L. et al. Liposome-mediatedNGF gene transfection following neuronal injury: potential therapeuticapplications. Gene Ther 6, 994-1005 (1999)). Hydrophobically modifiedsiRNAs have also been used as an alternative for cellular and braindelivery (Sah, Supra; Soutschek, J. et al. Therapeutic silencing of anendogenous gene by systemic administration of modified siRNAs. Nature432, 173-178 (2004); Cheng, K., Ye, Z., Guntaka, R. V. & Mahato, R. I.Enhanced hepatic uptake and bioactivity of type alpha1(I) collagen genepromoter-specific triplex-forming oligonucleotides after conjugationwith cholesterol. Journal of Pharmacology and Experimental Therapeutics317, 797-805 (2006); Byrne, M. et al. Novel Hydrophobically ModifiedAsymmetric RNAi Compounds (sd-rxRNA) Demonstrate Robust Efficacy in theEye. Journal of Ocular Pharmacology and Therapeutics 29, 855-864(2013)), and some of these compounds have even made it to clinic, butensuring both chemical stability and minimal toxicity while maximizingdelivery remains a difficult task. Current hurdles in RNAi technologylimit its ability to be used for both functional genomics studies andtherapeutics, providing an opportunity for improvement to their designas it applies to the area of neuroscience both in vitro and in vivo.

SUMMARY

Accordingly, provided herein are novel huntingtin target sequences. Alsoprovided herein are novel RNA molecules (e.g., siRNAs) that target thenovel huntingtin target sequences. Said novel RNA molecules (e.g.,siRNAs) demonstrate efficacy and potency in both primary neurons invitro, and in vivo in mouse brain subsequent to a single, low doseinjection.

In one aspect, an RNA molecule is provided that is between 15 and 30bases in length or between 15 and 35 bases in length, comprising aregion of complementarity which is substantially complementary to 5′CAGUAAAGAGAUUAA 3′ (SEQ ID NO:1).

In certain embodiments, the RNA molecule is single stranded (ss) RNA ordouble stranded (ds) RNA. In certain embodiments, the dsRNA comprises asense strand and an antisense strand, wherein the antisense strandcomprises the region of complementarity which is substantiallycomplementary to 5′ CAGUAAAGAGAUUAA 3′ (SEQ ID NO:1).

In certain embodiments, the dsRNA is between 30 and 35 base pairs inlength. In certain embodiments the region of complementarity iscomplementary to at least 10, 11, 12 or 13 contiguous nucleotides of SEQID NO:1. In certain embodiments, the region of complementarity containsno more than 3 mismatches with SEQ ID NO:1. In certain embodiments, theregion of complementarity is fully complementary to SEQ ID NO:1.

In certain embodiments, the dsRNA is blunt-ended. In certainembodiments, the dsRNA comprises at least one single stranded nucleotideoverhang. In certain embodiments, the dsRNA comprises naturallyoccurring nucleotides.

In certain embodiments, the dsRNA comprises at least one modifiednucleotide. In certain embodiments, the modified nucleotide is chosenfrom the group of: a 2′-O-methyl modified nucleotide, a nucleotidecomprising a 5′phosphorothioate group, and a terminal nucleotide linkedto a cholesteryl derivative or dodecanoic acid bisdecylamide group. Incertain embodiments, the modified nucleotide is chosen from the groupof: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modifiednucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modifiednucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, aphosphoramidate, and a non-natural base comprising nucleotide. Incertain embodiments, the dsRNA comprises at least one 2′-O-methylmodified nucleotide and at least one nucleotide comprising a5′phosphorothioate group.

In certain embodiments, the RNA molecule comprises a 5′ end, a 3′ endand has complementarity to a target, wherein: (1) the RNA moleculecomprises alternating 2′-methoxy-ribonucleotides and2′-fluoro-ribonucleotides; (2) the nucleotides at positions 2 and 14from the 5′ end are not 2′-methoxy-ribonucleotides; (3) the nucleotidesare connected via phosphodiester or phosphorothioate linkages; and (4)the nucleotides at positions 1-6 from the 3′ end, or positions 1-7 fromthe 3′ end, are connected to adjacent nucleotides via phosphorothioatelinkages.

In certain embodiments, the dsRNA has a 5′ end, a 3′ end andcomplementarity to a target, and comprises a first oligonucleotide and asecond oligonucleotide, wherein: (1) the first oligonucleotide comprisesa sequence set forth as SEQ ID NO:1; (2) a portion of the firstoligonucleotide is complementary to a portion of the secondoligonucleotide; (3) the second oligonucleotide comprises alternating2′-methoxy-ribonucleotides and 2′-fluoro-ribonucleotides; (4) thenucleotides at positions 2 and 14 from the 3′ end of the secondoligonucleotide are 2′-methoxy-ribonucleotides; and (5) the nucleotidesof the second oligonucleotide are connected via phosphodiester orphosphorothioate linkages.

In certain embodiments, the second oligonucleotide is linked to ahydrophobic molecule at the 3′ end of the second oligonucleotide. Incertain embodiments, the linkage between the second oligonucleotide andthe hydrophobic molecule comprises polyethylene glycol or triethyleneglycol. In certain embodiments, the nucleotides at positions 1 and 2from the 3′ end of second oligonucleotide are connected to adjacentnucleotides via phosphorothioate linkages. In certain embodiments, thenucleotides at positions 1 and 2 from the 3′ end of secondoligonucleotide, and the nucleotides at positions 1 and 2 from the 5′end of second oligonucleotide, are connected to adjacent ribonucleotidesvia phosphorothioate linkages.

In certain aspects, a pharmaceutical composition for inhibiting theexpression of the HTT gene in an organism, comprising a dsRNA and apharmaceutically acceptable carrier is provided. The dsRNA comprises asense strand and an antisense strand. The dsRNA is between 15 and 35base pairs in length and the antisense strand comprises a region ofcomplementarity which is substantially complementary to 5′CAGUAAAGAGAUUAA 3′ (SEQ ID NO:1).

In certain embodiments, the dsRNA comprises a cholesterol moiety.

In certain aspects, a method for inhibiting expression of HTT gene in acell is provided. The method includes the steps of introducing into thecell a double-stranded ribonucleic acid (dsRNA) comprising a sensestrand and an antisense strand, the dsRNA is between 15 and 35 basepairs in length and the antisense strand comprises a region ofcomplementarity which is substantially complementary to 5′CAGUAAAGAGAUUAA 3′ (SEQ ID NO:1), and maintaining the cell produced instep (a) for a time sufficient to obtain degradation of the mRNAtranscript of the HTT gene, thereby inhibiting expression of the HTTgene in the cell.

In certain aspects, a method of treating or managing Huntington'sdisease comprising administering to a patient in need of such treatmentor management a therapeutically effective amount of a dsRNA is provided.The dsRNA comprises a sense strand and an antisense strand, and isbetween 15 and 35 base pairs in length, and the antisense strandcomprises a region of complementarity which is substantiallycomplementary to 5′ CAGUAAAGAGAUUAA 3′ (SEQ ID NO:1).

In certain embodiments, the dsRNA is administered to the brain of thepatient. In certain embodiments, the dsRNA is administered by any ofintrastriatal, intracerebroventricular and/or intrathecal infusionand/or pump. In certain embodiments, administering the dsRNA to thebrain causes a decrease in HTT gene mRNA in the striatum. In certainembodiments, administering the dsRNA to the brain causes a decrease inHTT gene mRNA in the cortex.

In certain aspects, a vector for inhibiting the expression of HTT genein a cell is provided. The vector comprising a regulatory sequenceoperably linked to a nucleotide sequence that encodes an RNA moleculesubstantially complementary to 5′ CAGUAAAGAGAUUAA 3′ (SEQ ID NO:1),wherein said RNA molecule is between 15 and 35 bases in length, andwherein said RNA molecule, upon contact with a cell expressing said HTTgene, inhibits the expression of said HTT gene by at least 20%.

In certain embodiments, the RNA molecule is ssRNA or dsRNA. In certainembodiments, the dsRNA comprises a sense strand and an antisense strand,wherein the antisense strand comprises the region of complementaritywhich is substantially complementary to 5′ CAGUAAAGAGAUUAA 3′ (SEQ IDNO:1).

In certain aspects, a cell comprising a vector for inhibiting theexpression of HTT gene in a cell is provided. The vector comprising aregulatory sequence operably linked to a nucleotide sequence thatencodes an RNA molecule substantially complementary to 5′CAGUAAAGAGAUUAA 3′ (SEQ ID NO:1), wherein said RNA molecule is between15 and 35 bases in length, and wherein said RNA molecule, upon contactwith a cell expressing said HTT gene, inhibits the expression of saidHTT gene by at least 20%.

In certain embodiments, the RNA molecule is ssRNA or dsRNA. In certainembodiments, the dsRNA comprises a sense strand and an antisense strand,wherein the antisense strand comprises the region of complementaritywhich is substantially complementary to 5′ CAGUAAAGAGAUUAA 3′ (SEQ IDNO:1).

In one aspect, an RNA molecule is provided that is between 15 and 35bases in length, comprising a region of complementarity which issubstantially complementary to 5′ AUAUCAGUAAAGAGA 3′ (SEQ ID NO:2) or 5′CUCAGGAUUUAAAAU 3′ (SEQ ID NO:3).

In certain embodiments, the RNA molecule is single stranded (ss) RNA ordouble stranded (ds) RNA. In certain embodiments, the dsRNA comprises asense strand and an antisense strand, wherein the antisense strandcomprises the region of complementarity which is substantiallycomplementary to 5′ CAGUAAAGAGAUUAA 3′ (SEQ ID NO:1).

In certain embodiments, the dsRNA is between 30 and 35 base pairs inlength. In certain embodiments the region of complementarity iscomplementary to at least 10, 11, 12 or 13 contiguous nucleotides of SEQID NO:2 or 3. In certain embodiments, the region of complementaritycontains no more than 3 mismatches with SEQ ID NO: 1 . In certainembodiments, the region of complementarity is fully complementary to SEQID NO:2 or 3.

In certain embodiments, the dsRNA is blunt-ended. In certainembodiments, the dsRNA comprises at least one single stranded nucleotideoverhang. In certain embodiments, the dsRNA comprises naturallyoccurring nucleotides.

In certain embodiments, the dsRNA comprises at least one modifiednucleotide. In certain embodiments, the modified nucleotide is chosenfrom the group of: a 2′-O-methyl modified nucleotide, a nucleotidecomprising a 5′phosphorothioate group, and a terminal nucleotide linkedto a cholesteryl derivative or dodecanoic acid bisdecylamide group. Incertain embodiments, the modified nucleotide is chosen from the groupof: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modifiednucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modifiednucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, aphosphoramidate, and a non-natural base comprising nucleotide. Incertain embodiments, the dsRNA comprises at least one 2′-0-methylmodified nucleotide and at least one nucleotide comprising a5′phosphorothioate group.

In certain embodiments, the RNA molecule comprises a 5′ end, a 3′ endand has complementarity to a target, wherein: (1) the RNA moleculecomprises alternating 2′-methoxy-ribonucleotides and2′-fluoro-ribonucleotides; (2) the nucleotides at positions 2 and 14from the 5′ end are not 2′-methoxy-ribonucleotides; (3) the nucleotidesare connected via phosphodiester or phosphorothioate linkages; and (4)the nucleotides at positions 1-6 from the 3′ end, or positions 1-7 fromthe 3′ end, are connected to adjacent nucleotides via phosphorothioatelinkages.

In certain embodiments, the dsRNA has a 5′ end, a 3′ end andcomplementarity to a target, and comprises a first oligonucleotide and asecond oligonucleotide, wherein: (1) the first oligonucleotide comprisesa sequence set forth as SEQ ID NO:1; (2) a portion of the firstoligonucleotide is complementary to a portion of the secondoligonucleotide; (3) the second oligonucleotide comprises alternating2′-methoxy-ribonucleotides and 2′-fluoro-ribonucleotides; (4) thenucleotides at positions 2 and 14 from the 3′ end of the secondoligonucleotide are 2′-methoxy-ribonucleotides; and (5) the nucleotidesof the second oligonucleotide are connected via phosphodiester orphosphorothioate linkages.

In certain embodiments, the second oligonucleotide is linked to ahydrophobic molecule at the 3′ end of the second oligonucleotide. Incertain embodiments, the linkage between the second oligonucleotide andthe hydrophobic molecule comprises polyethylene glycol or triethyleneglycol. In certain embodiments, the nucleotides at positions 1 and 2from the 3′ end of second oligonucleotide are connected to adjacentnucleotides via phosphorothioate linkages. In certain embodiments, thenucleotides at positions 1 and 2 from the 3′ end of secondoligonucleotide, and the nucleotides at positions 1 and 2 from the 5′end of second oligonucleotide, are connected to adjacent ribonucleotidesvia phosphorothioate linkages.

In certain aspects, a pharmaceutical composition for inhibiting theexpression of the HTT gene in an organism, comprising a dsRNA and apharmaceutically acceptable carrier is provided. The dsRNA comprises asense strand and an antisense strand. The dsRNA is between 15 and 35base pairs in length and the antisense strand comprises a region ofcomplementarity which is substantially complementary to 5′AUAUCAGUAAAGAGA 3′ (SEQ ID NO:2) or 5′ CUCAGGAUUUAAAAU 3′ (SEQ ID NO:3).

In certain embodiments, the dsRNA comprises a cholesterol moiety.

In certain aspects, a method for inhibiting expression of HTT gene in acell is provided. The method includes the steps of introducing into thecell a double-stranded ribonucleic acid (dsRNA) comprising a sensestrand and an antisense strand, the dsRNA is between 15 and 35 basepairs in length and the antisense strand comprises a region ofcomplementarity which is substantially complementary to 5′AUAUCAGUAAAGAGA 3′ (SEQ ID NO:2) or 5′ CUCAGGAUUUAAAAU 3′ (SEQ ID NO:3),and maintaining the cell produced in step (a) for a time sufficient toobtain degradation of the mRNA transcript of the HTT gene, therebyinhibiting expression of the HTT gene in the cell.

In certain aspects, a method of treating or managing Huntington'sdisease comprising administering to a patient in need of such treatmentor management a therapeutically effective amount of a dsRNA is provided.The dsRNA comprises a sense strand and an antisense strand, and isbetween 15 and 35 base pairs in length, and the antisense strandcomprises a region of complementarity which is substantiallycomplementary to 5′ AUAUCAGUAAAGAGA 3′ (SEQ ID NO:2) or 5′CUCAGGAUUUAAAAU 3′ (SEQ ID NO:3).

In certain embodiments, the dsRNA is administered to the brain of thepatient. In certain embodiments, the dsRNA is administered byintrastriatal infusion. In certain embodiments, administering the dsRNAto the brain causes a decrease in HTT gene mRNA in the striatum. Incertain embodiments, administering the dsRNA to the brain causes adecrease in HTT gene mRNA in the cortex.

In certain aspects, a vector for inhibiting the expression of HTT genein a cell is provided. The vector comprising a regulatory sequenceoperably linked to a nucleotide sequence that encodes an RNA moleculesubstantially complementary to 5′ AUAUCAGUAAAGAGA 3′ (SEQ ID NO:2) or 5′CUCAGGAUUUAAAAU 3′ (SEQ ID NO:3), wherein said RNA molecule is between15 and 35 bases in length, and wherein said RNA molecule, upon contactwith a cell expressing said HTT gene, inhibits the expression of saidHTT gene by at least 20%.

In certain embodiments, the RNA molecule is ssRNA or dsRNA. In certainembodiments, the dsRNA comprises a sense strand and an antisense strand,wherein the antisense strand comprises the region of complementaritywhich is substantially complementary to 5′ AUAUCAGUAAAGAGA 3′ (SEQ IDNO:2) or 5′ CUCAGGAUUUAAAAU 3′ (SEQ ID NO:3).

In certain aspects, a cell comprising a vector for inhibiting theexpression of HTT gene in a cell is provided. The vector comprising aregulatory sequence operably linked to a nucleotide sequence thatencodes an RNA molecule substantially complementary to 5′AUAUCAGUAAAGAGA 3′ (SEQ ID NO:2) or 5′ CUCAGGAUUUAAAAU 3′ (SEQ ID NO:3),wherein said RNA molecule is between 15 and 35 bases in length, andwherein said RNA molecule, upon contact with a cell expressing said HTTgene, inhibits the expression of said HTT gene by at least 20%.

In certain embodiments, the RNA molecule is ssRNA or dsRNA. In certainembodiments, the dsRNA comprises a sense strand and an antisense strand,wherein the antisense strand comprises the region of complementaritywhich is substantially complementary to 5′ AUAUCAGUAAAGAGA 3′ (SEQ IDNO:2) or 5′ CUCAGGAUUUAAAAU 3′ (SEQ ID NO:3).

In certain aspects, an RNA molecule that is between 15 and 35 bases inlength is provided. The RNA molecule comprises a region ofcomplementarity which is substantially complementary to 5′CAGUAAAGAGAUUAA 3′ (SEQ ID NO:1), 5′ AUAUCAGUAAAGAGA 3′ (SEQ ID NO:2) or5′ CUCAGGAUUUAAAAU 3′ (SEQ ID NO:3), and the RNA molecule targets a 3′untranslated region (UTR) of HTT gene short mRNA.

The 3′ UTR of the HTT gene short mRNA is as follows:

(SEQ ID NO: 4) AGCGCCAUGGUGGGAGAGACUGUGAGGCGGCAGCUGGGGCCGGAGCCUUUGGAAGUCUGCGCCCUUGUGCC CUGCCUCCACCGAGCCAGCUUGGUCCCUAUGGGCUUCCGCACAUGCCGCGGGCGGCCAGGCAACGUGCGU GUCUCUGCCAUGUGGCAGAAGUGCUCUUUGUGGCAGUGGCCAGGCAGGGAGUGUCUGCAGUCCUGGUGGG GCUGAGCCUGAGGCCUUCCAGAAAGCAGGAGCAGCUGUGCUGCACCCCAUGUGGGUGACCAGGUCCUUUC UCCUGAUAGUCACCUGCUGGUUGUUGCCAGGUUGCAGCUGCUCUUGCAUCUGGGCCAGAAGUCCUCCCUC CUGCAGGCUGGCUGUUGGCCCCUCUGCUGUCCUGCAGUAGAAGGUGCCGUGAGCAGGCUUUGGGAACACU GGCCUGGGUCUCCCUGGUGGGGUGUGCAUGCCACGCCCCGUGUCUGGAUGCACAGAUGCCAUGGCCUGUG CUGGGCCAGUGGCUGGGGGUGCUAGACACCCGGCACCAUUCUCCCUUCUCUCUUUUCUUCUCAGGAUUUA AAAUUUAAUUAUAUCAGUAAAGAGAUUAAUUUUAACGUAACUCUUUCUAUGCCCGUGUA

In certain embodiments, the RNA molecule is ssRNA or dsRNA. In certainembodiments, the dsRNA comprises a sense strand and an antisense strand,wherein the antisense strand comprises the region of complementaritywhich is substantially complementary to 5′ CAGUAAAGAGAUUAA 3′ (SEQ IDNO:1), 5′ AUAUCAGUAAAGAGA 3′ (SEQ ID NO:2) or 5′ CUCAGGAUUUAAAAU 3′ (SEQID NO:3).

In certain aspects, a dsRNA molecule that is between 15 and 35 bases inlength, comprising a region of complementarity which is substantiallycomplementary to 5′ CAGUAAAGAGAUUAA 3′ (SEQ ID NO:1), 5′ AUAUCAGUAAAGAGA3′ (SEQ ID NO:2) or 5′ CUCAGGAUUUAAAAU 3′ (SEQ ID NO:3), wherein the RNAmolecule targets an HTT mRNA and comprises at least one modifiednucleotide is provided. In certain embodiments, the modified nucleotideis a terminal nucleotide linked to a phosphatidylcholine derivative.

In certain aspects, a di-branched RNA compound comprising two RNAmolecules that are between 15 and 35 bases in length, comprising aregion of complementarity which is substantially complementary to 5′CAGUAAAGAGAUUAA 3′ (SEQ ID NO:1), 5′ AUAUCAGUAAAGAGA 3′ (SEQ ID NO:2) or5′ CUCAGGAUUUAAAAU 3′ (SEQ ID NO:3), wherein the two RNA molecules areconnected to one another by one or more moieties independently selectedfrom a linker, a spacer and a branching point, is provided.

In any of the aspects described herein, the RNA molecule is an antisensemolecule (e.g., ASO) or a GAPMER molecule. In certain embodiments, theantisense molecule enhances degradation of the region ofcomplementarity. In certain embodiments, the degradation is nucleasedegradation (e.g., RNase H).

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings. The patent or application file contains at least one drawingexecuted in color. Copies of this patent or patent applicationpublication with color drawing(s) will be provided by the Office uponrequest and payment of the necessary fee.

FIGS. 1A-1B depict hydrophobic siRNA structural and chemical compositionand efficient internalization in primary cortical neurons. A) Schematicof the hydrophobically modified and stabilized siRNAs (hsiRNAs) B)Cy3-HTT10150 hsiRNA (red), 0.5 μM, was added to primary corticalneurons. Imaged on Zeiss confocal microscope, 63×, nuclei stained withHoechst dye (blue).

FIGS. 2A-2C depict a systematic screening of unformulated hsiRNAstargeting huntingtin mRNA plotted as a line graph (A) or a bar graphs(B) and (C). A panel of 94 hsiRNAs were added to HeLa cells at 1.5 μM.Level of huntingtin mRNA was measured using QUANTIGENE (Affymetrix) at72 hours normalized to housekeeping gene, PPIB (cyclophilin B), andpresented as percent of untreated control (n=3, mean+/−SD).UNT—untreated cells, NTC—non-targeting control. Active compounds (red)were selected for further analysis.

FIGS. 3A-3C depict concentration-dependent silencing of huntingtin mRNAby HTT10150, in both passive (A) and lipid-mediated delivery (B).Chemical modifications enable passive uptake without negative impact onsiRNA RISC (RNA Induced Silencing Complex) entry. HeLa cells wereincubated with modified (containing both hydrophobic and base chemicalmodifications) or unmodified HTT10150 at concentrations shown in theabsence (A) and presence (B) of RNAIMAX. Level of huntingtin mRNA wasmeasured using QUANTIGENE (Affymetrix) at 72 hours normalized tohousekeeping gene, PPIB (cyclophillin B), and presented as percent ofuntreated control (n=3, mean+/−SD). UNT—untreated cells. IC50 valuescalculated as described herein. (C) Is a table summarizing theseresults.

FIGS. 4A-4B graphically depict concentration-dependent silencing ofhuntingtin mRNA and protein by HTT10150 in primary neurons (passiveuptake). Primary neurons were incubated with HTT10150 at concentrationsshown. Level of huntingtin mRNA was measured using QUANTIGENE(Affymetrix) normalized to housekeeping gene, PPIB (cyclophillin B), andpresented as percent of untreated control (n=3, mean+/−SD).UNT—untreated cells. A) In primary cortical and striatal neurons, 1week. B) Huntingtin protein levels after one week incubation withHTT10150 were detected by western blot and normalized to β-Tubulin.

FIGS. 5A-5H depict a single intrastriatal injection of HTT10150 islocalized to neurons and fiber tracts ipsilateral to the injection siteafter 24 hours. 1 nmol CY3-HTT10150 (Red) was unilaterally injected intothe striatum of WT (FVBNj) mice. Brains were collected after 24 hours,paraffin imbedded and sectioned and sectioned. (FIG. 5A) Tiled image ofcoronal brain section (16×). Majority of HTT10150 was localized at siteof injection with sharp gradient of diffusion. (FIG. 5B) Tiled image ofsagittal brain section (16×), injected side. (FIG. 5C) Image of coronalbrain section (40×), non-injected side. (FIG. 5D) Image of coronal brainsection (40×), injected side. (FIG. 5E, FIG. 5G) NueN stained neuronsfrom non-injected side (60×). (FIG. 5F, FIG. 5H) NueN stained neuronsfrom injected side (60×).

FIG. 6 graphically depicts evaluation of HTT10150 efficacy in vivo.HTT10150 was unilaterally injected into the striatum of WT (FVB) mice (2μl). Mice were sacrificed at 120 hours. Brains were sliced into 300 μmsections and six—2 mm punch biopsies of the striatum were collected fromboth Ipsilateral and Contralateral sides. Level of huntingtin mRNA wasmeasured using QUANTIGENE (Affymetrix) normalized to housekeeping gene,PPIB (cyclophillin B), and presented as percent of untreated control(n=24, mean+/−SEM, 8 animals, 3 biopsies per region).

FIGS. 7A-7E depict that HTT10150 shows no toxicity in DARPP-32 positiveneurons around the site of injection. HTT10150 was unilaterally injectedinto the striatum of WT (FVB) mice. Brains were collected after 5 daysfixed, sectioned, and stained with antibodies against DARPP-32 (A-D).Representative image of striatum after injection of ACSF, full brainscan and 60× magnification (A, B) or 12.5 μg HTT10150, full brain scanand 60× magnification (C, D). Quantification of DARPP-32 positiveneurons (E) (n=3 animals, mean+/−SD).

FIG. 8 depicts target sequences (SEQ ID NOS 1045-1057, 3, 2, 1 and1061-1139, respectively, in order of appearance), modifiedoligonucleotides (“Sense Strand” sequences disclosed as SEQ ID NOS1140-1231 and “Antisense Strand” sequences disclosed as SEQ ID NOS1232-1326, all respectively, in order of appearance) and their efficacyaccording to certain embodiments.

FIG. 9 depicts efficient uptake and internalization of hsiRNA in primarycortical neurons over time. Cy3-HTT10150 hsiRNA (red), 0.5 μM, was addedto primary cortical neurons. Imaged on Zeiss confocal microscope, 63×,nuclei stained with Hoechst dye (blue).

FIGS. 10A-10B graphically depict concentration-dependent silencing ofhuntingtin mRNA by HTT10150 in HeLa cells. Level of huntingtin mRNA wasmeasured using QUANTIGENE (Affymetrix) at 72 hours normalized tohousekeeping gene, PPIB (cyclophillin B), and presented as percent ofuntreated control (n=3, mean+/−SD). UNT—untreated cells,NTC—non-targeting control. A) Dose response of 16 active sequences inpassive uptake (no formulation). B) Dose response of eight selectedsequences in lipid-mediated uptake (using Invitrogen LIPOFECTAMINERNAIMAX Transfection Reagent). Dose response data was fitted usingGraphPad Prism 6.03.

FIGS. 11A-11B graphically depict huntingtin mRNA levels. A) Cellviability was tested using ALAMAR BLUE (Life Technologies) afterincubation of HTT10150 and NTC with primary cortical neurons for 72hours and one week. B) Primary cortical neurons were incubated withthree HTT hsiRNA sequences HTT10150, HTT10146, and HTT1215 atconcentrations shown. Level of huntingtin mRNA was measured usingQUANTIGENE (Affymetrix) normalized to housekeeping gene, PPIB(cyclophillin B), and presented as percent of untreated control (n=3,mean+/−SD). UNT—untreated cells.

FIGS. 12A-12B graphically depict concentration-dependent silencing ofhuntingtin mRNA by HTT10150 in primary neurons (passive uptake). Primaryneurons were incubated with HTT10150 at concentrations shown. Level ofhuntingtin mRNA was measured using QUANTIGENE (Affymetrix) normalized tohousekeeping gene, PPIB (cyclophillin B), and presented as percent ofuntreated control (n=3, mean+/−SD). UNT—untreated cells. A) For 72 hoursand 1 week. B) For 1, 2 and 3 weeks.

FIG. 13 graphically depicts efficacy of hsiRNA against cyclophilin B(PPIB) in primary cortical neurons. Primary neurons were incubated withhsiRNA targeting PPIB at concentrations shown. Level of PPIB mRNA wasmeasured using QUANTIGENE (Affymetrix) normalized to housekeeping gene,HTT and presented as percent of untreated control (n=3, mean+/−SD).UNT—untreated cells for 1 week.

FIG. 14 depicts representative Western blots of Htt reduction in primarycortical neurons. Primary cortical neurons were cultured from fiveindividual pups (#1-5) and incubated with HTT10150 at concentrationsshown for one week. Huntingtin protein levels were detected by Westernblot using antibody AB1.

FIGS. 15A-15B graphically depict evaluation of HTT10150 efficacy invivo. A) HTT10150 was unilaterally injected into the striatum of WT(FVB) mice (2 μl). Mice were sacrificed at 120 hours. Brains were slicedinto 300 μm sections and six 2 mm punch biopsies of the striatum werecollected from both ipsilateral and contralateral sides. Level ofhuntingtin mRNA was measured using QUANTIGENE (Affymetrix) normalized tohousekeeping gene, PPIB (cyclophilin B), and presented as percent ofuntreated control (n=8 animals, mean+/−SD). B) Quantification ofhuntingtin protein silencing by Western blot.

FIG. 16 graphically depicts evaluation of HTT10150 cytotoxicity in vivo.DARPP32 neuronal marker was minimally affected by HTT10150 injection,indicating no major impact on neuronal health. HTT10150 was unilaterallyinjected into the striatum of wild-type (FVB) mice at doses shown. Micewere sacrificed at 120 hours. Brains were sliced into 300 μm sectionsand six punch biopsies (2 mm) of the striatum were collected from bothipsilateral and contralateral sides. Level of DARPP32 mRNA expressionwas measured using QUANTIGENE (Affymetrix) normalized to housekeepinggene, PPIB (cyclophilin B), and presented as percent of untreatedcontrol (n=24, mean+/−SD).

FIGS. 17A-17C depict that HTT10150 showed a two-fold increase inmicroglial activation at the site of injection. HTT10150 wasunilaterally injected into the striatum of WT (FVB) mice. Brains werecollected after 6 hours (b) and 5 days (a and c) fixed, sectioned, andstained with antibodies against IBA-1. (A) Representative images ofactivated (black arrowhead) and resting (open arrowhead) after injectionof 1 nmol HTT10150 and ACSF 5 days post injection. 40× magnification.(B) Quantification of activated and resting microglia 6 hrspost-injection of ACSF (n=6) and lnmol HTT10150 (n=3). (C)Quantification of activated and resting microglia 5 days post-injectionof ACSF (n=4) and lnmol HTT10150 (n=3).

FIGS. 18A-18C depict that HTT10150 showed limited toxicity at the siteof injection at the 25 μg dose. HTT10150 was unilaterally injected intothe striatum of WT (FVB) mice. Brains were collected after 5 days fixed,sectioned, and stained with antibodies against DARPP-32. Representativeimage of striatum after injection of 25 μg, full brain scan (A), 10×magnification at injections site (B), 20× magnification at injectionsite (C), and 60× magnification.

FIG. 19 depicts that HTT10150 showed no toxicity to Darpp32 positiveneurons at lower concentrations. HTT10150 was unilaterally injected intothe striatum of WT (FVB) mice. Brains were collected after 5 days fixed,sectioned, and stained with antibodies against DARPP-32. Representativeimage of striatum after injection of 25 μg, 12.5 μg, and ACSF (20×magnification) ipsilateral and contralateral to the site of injection.

FIGS. 20A-20B depict that HTT10150 caused a slight increase in totalresting microglia 5 days post injection. HTT10150 was unilaterallyinjected into the striatum of WT (FVB) mice. Brains were collected after6 hours and 5 days fixed, sectioned, and stained with antibodies againstIBA-1. Quantification of total microglia 6 hrs (A) and 5 days (B)post-injection of ACSF (n=6, A) (n=4, B) and 12.5 μg HTT10150 (n=3, A,B).

FIG. 21 depicts additional target sequences (SEQ ID NOS 5-212,respectively, in order of columns) along with chemical modifications andstructural scaffolds according to certain embodiments of the invention(“Sense Naked” sequences disclosed as SEQ ID NOS 213-316, “AnstisenseNaked” sequences disclosed as SEQ ID NOS 317-420, “Sense Strand (P0)”sequences disclosed as SEQ ID NOS 421-524, “Antisense Strand (P0)”sequences disclosed as SEQ ID NOS 525-628, “Sense Strand (P1)” sequencesdisclosed as SEQ ID NOS 629-732, “Antisense Strand (P1)” sequencesdisclosed as SEQ ID NOS 733-836, “Sense Strand (P2)” sequences disclosedas SEQ ID NOS 837-940, and “Antisense Strand (P2)” sequences disclosedas SEQ ID NOS 941-1044, all respectively, in order of columns).

FIG. 22 depicts hsiRNA^(HTT) efficacy in primary cortical neurons (cellviability) after one week using QUANTIGENE and ALAMAR BLUE.NTC=non-targeting control.

FIG. 23 depicts HTT hsiRNA efficacy in wild-type primary striatalneurons and primary cortical neurons after one week using QUANTIGENE.NTC=non-targeting control.

FIG. 24 depicts HTT hsiRNA efficacy in primary neurons (duration ofeffect) from one to three weeks post-treatment via passive uptake. HTTexpression was normalized to PPIB. Data is shown is an approximatepercentage of non-targeting control. UNT=untreated.

FIG. 25 graphically depicts that hsiRNA^(HTT) but not LNA-GAPMERexhibits a silencing plateau in cortical neurons after 72 hours usingQUANTIGENE. N=3.

FIG. 26 shows intracellular localization of htt and ppib in primarycortical neurons using RNA-SCOPE. Htt mRNA, red; ppib mRNA, green;nuclei (DAPI), blue.

FIG. 27 validates in neurons an htt detection probe set, affirmingspecificity.

FIG. 28 validates in neurons an htt detection probe set, showing thatthe signal is not intron-specific (validated for intron 60-61).

FIG. 29 depicts that htt mRNA nuclear localization is specific toneurons only. Left panel depicts primary neurons; ppib mRNA, green; httmRNA, red, nuclei, blue.

FIG. 30 depicts that hsiRNA^(HTT) treatment of cortical neuronspreferentially eliminates cytoplasmic htt mRNA. Ppib mRNA, green; httmRNA, red; nuclei, blue. Top panel: non-treated. Bottom panel, treatedwith 1.5 μM hsiRNA^(HTT) for three days.

FIG. 31 graphically depicts that hsiRNA^(HTT) treatment of corticalneurons preferentially eliminates cytoplasmic htt mRNA.

FIG. 32 depicts a Western blot showing HTT protein silencing inwild-type primary cortical neurons. hsiRNA htt-10150; NTC=non-targetingcontrol, 1 week.

FIG. 33 graphically depicts the results of HTT10150 direct injection. Noeffects on neuronal viability were observed.

FIG. 34 depicts toxicity adjacent to the injection site followingcholesterol-hsiRNA administration.

FIGS. 35A-35C show that partially modified hsiRNAs exhibit a shortduration of effect and no systemic exposure.

FIGS. 36A-36C depict full metabolic stabilization of hsiRNAs.

FIGS. 37A-37C show that full metabolic stabilization does not interferewith RISC entry of hsiRNAs.

FIGS. 38A-38E depict fully metabolically stabilized hsiRNA (FM-hsiRNA)enhancement of local delivery and distribution.

FIGS. 39A-39B depict enhanced potency and duration of effect mediated byFM-hsiRNA.

FIG. 40A-40B characterizes neuroactive, naturally occurring lipids ashsiRNA bioconjugates.

FIG. 41 depicts that hsiRNA hydrophobicity directly correlates withbrain distribution and retention. Intrastriatal injection, 12.5 μg (0.5mg/kg), t=24 hours, FVB/NJ mice (n=2).

FIG. 42 depicts docosahexaenoic acid (DHA) hsiRNA synthesis.

FIG. 43 depicts internalization of DHA-hsiRNA and chol-hsiRNA intoprimary cortical neurons. Uptake: 0.5 μM Cy3-DHA-hsiRNA (red), DAPI(blue).

FIG. 44 depicts co-localization of DHA-hsiRNA with neurons andastrocytes. Intrastriatal injection, 12.5 μg (0.5 mg/kg), t=24 hours,FVB/NJ mice (n=2).

FIG. 45 depicts localization of DHA-hsiRNA to the perinuclear region instriatal neurons, while chol-hsiRNA is undetectable. Intrastriatalinjection, 12.5 μg (0.5 mg/kg), t=24 hours, FVB/NJ mice (n=2).

FIG. 46 depicts co-localization of DHA-hsiRNA with neurons andastrocytes in the cortex following a single intrastriatal injection.Intrastriatal injection, 12.5 μg (0.5 mg/kg), t=24 hours, FVB/NJ mice(n=2).

FIG. 47 depicts localization of DHA-hsiRNA to the perinuclear region incortical neurons, while chol-hsiRNA is undetectable.

FIG. 48 depicts robust silencing efficiency of DHA-hsiRNA in thestriatum and cortex. Intrastriatal injection, 6-25 μg (0.25-1 mg/kg),t=5 days, FVB/NJ mice (n=8).

FIG. 49 depicts the duration of effect and recovery in the striatumfollowing a single intrastriatal dose of DHA-hsiRNA.

FIG. 50 depicts a pilot safety study showing that DHA-siRNA does notaffect striatal neuronal integrity at greater than 20-fold over theefficacious dose.

FIG. 51 depicts a pilot safety study showing that DHA-siRNA causesminimal striatal microglial activation at greater than 20-fold over theefficacious dose.

FIG. 52 depicts perinuclear localization caused by oligonucleotidechemistry.

FIG. 53 depicts intra-nuclear foci distribution caused byoligonucleotide chemistry.

FIG. 54 shows that the degree of htt mRNA striatal silencing is effectedby oligonucleotide cellular localization.

FIG. 55 depicts targeted glial delivery.

FIG. 56 depicts targeted neuronal delivery.

FIG. 57 shows that DHA-hsiRNA efficiently distributes throughout thebrain and silences genes in both the striatum and the cortex.Intrastriatal injection, 12.5 μg (0.5 mg/kg), t=24 hours, FVB/NJ mice(n=2).

FIG. 58 shows hsiRNA efficacy in wild-type primary hippocampal neuronsand Q140 primary hippocampal neurons. 16% gel.

FIG. 59 graphically depicts hsiRNA efficacy in wild-type primaryhippocampal neurons and Q140 primary hippocampal neurons.

FIG. 60 shows hsiRNA efficacy in wild-type primary hippocampal neuronsand Q140 primary hippocampal neurons. 7.5% gel.

FIG. 61 shows that each of PC-DHA-hsiRNA and chol-hsiRNA silence mutantand wild-type htt mRNA.

FIG. 62 describes three classes of hsiRNA chemistries: DHA-hsiRNA,PC-DHA-hsiRNA and chol-hsiRNA.

FIGS. 63A-63B graphically depict enhanced potency of PC-DHA-hsiRNArelative to DHA-hsiRNA in cortical primary neurons. 1 week, analyzed byQUANTIGENE, data normalized to PPIB.

FIG. 64 illustrates that chol-hsiRNA has a more effective chemistry forgene modulation in primary cortical neurons relative to PC-DHA-hsiRNAand DHA-hsiRNA. 1 week, analyzed by QUANTIGENE, data normalized to PPIB.

FIG. 65 shows that PC-DHA-hsiRNA shows better brain retention and widerdistribution that DHA-hsiRNA. Intrastriatal injections at either 2 or 10nmol, N=2, brains collected at 48 hours.

FIG. 66 shows approximately 80% silencing in mouse striatum after asingle IS injection PC-DHA-hsiRNA.

FIG. 67 shows approximately 60% silencing in mouse cortex after a singleIS injection PC-DHA-hsiRNA.

FIG. 68 depicts di-hsiRNA brain distribution after an CSF bolusinjection (250 μg), 48 hours.

FIG. 69 depicts distribution of di-hsiRNA after a single IS injection.

FIG. 70 depicts effect of branching on brain distribution.

FIG. 71 depicts a study design to assay in vivo gene silencing aftersingle IS injections of di-hsiRNA.

FIG. 72 depicts neuronal delivery of di-hsiRNA.

FIG. 73 depicts efficacy of di-hsiRNA in the striatum and cortex. ISinjection, 2 nmol di-hsiRNA, 1 week, QuantiGene 2.0.

FIG. 74 depicts uniform spinal cord distribution of di-hsiRNA.

FIG. 75 depicts htt mRNA silencing in the spinal cord afteradministration of a di-hsiRNA^(HTT) bolus. IT, 3 nmol, one week,QuantiGene.

FIG. 76A-76B depicts di-hsiRNA-mediated in vitro silencing in HeLa cellsand primary cortical neurons.

FIG. 77 depicts biodistribution of di-hsiRNA. Intrastriatal injection of2 nmol of Di-siRNA oligo (4 nmol of corresponding antisense strand). N=2mice per conjugate. Brains collected 48 hours later and stained withDAPI (nuclei, blue) and NeuN (neuronal marker, green). Image isrepresentative. Red-oligo.

FIG. 78 depicts biodistribution of di-hsiRNA. Intrastriatal injection of2 nmol of Di-siRNA oligo (4 nmol of corresponding antisense strand). N=2mice per conjugate. Brains collected 48 hours later and stained withDAPI (nuclei, blue) and NeuN (neuronal marker, green). Image isrepresentative. Red-oligo.

FIG. 79 depicts brain distribution of di-hsiRNA, TEG-azide, TEG andvitamin D after 48 hours. 2 nmole injected IS, N=2 mice per conjugate,brains collected 48 hours later.

FIG. 80 depicts the efficacy of vitamin D synthesis on htt mRNAexpression.

FIG. 81 depicts a chemical Formula of a compound provided herein. Fig.discloses SEQ ID NOS 1327, 1328, 1327, and 1328, respectively, in orderof appearance.

FIG. 82 depicts examples of internucleotide linkages of R³.

FIG. 83 depicts an embodiment of the chemical Formula of FIG. 81. Fig.discloses SEQ ID NOS 1327, 1328, 1327, and 1328, respectively, in orderof appearance.

FIG. 84 depicts a chemical Formula of a compound provided herein. Fig.discloses SEQ ID NOS 1327-1328, respectively, in order of appearance.

FIG. 85 depicts a chemical Formula of a compound provided herein. Fig.discloses SEQ ID NOS 1327-1328, respectively, in order of appearance.

FIG. 86 depicts an embodiment of the Y moiety of FIG. 84 or FIG. 85.Fig. discloses SEQ ID NOS 1327-1328, respectively, in order ofappearance.

FIG. 87 depicts a chemical Formula of a compound provided herein. Fig.discloses SEQ ID NOS 1327, 1328, 1327, and 1328, respectively, in orderof appearance.

FIG. 88 depicts an embodiment of the chemical Formula of FIG. 87. Fig.discloses SEQ ID NOS 1327, 1328, 1327, and 1328, respectively, in orderof appearance.

FIG. 89 depicts a chemical Formula of a compound provided herein. Fig.discloses SEQ ID NOS 1327-1328, respectively, in order of appearance.

FIG. 90 depicts an embodiment of the chemical Formula of FIG. 89. Fig.discloses SEQ ID NOS 1327-1328, respectively, in order of appearance.

FIGS. 91A-91D depict the development of fully metabolically stabilizedhsiRNAs (FM-hsiRNAs). (A) Schematics of partially and fully modifiedhsiRNAs. (B) hsiRNA and FM-hsiRNA have equal ability to enter RISC(HeLa, 72 hours). (C) Metabolically stable 5′-E-VP is as active as 5′-P.(D) 5′-E-VP enables sustained delivery to distant tissues (7 days postinjection, PNA assay).

FIG. 92 depicts that the evolution of chemistry enabled widedistribution of hsiRNA in mouse brain after a bolus CSF (ICV) infusion.Images of sagittal sections (left panels) from 48 hours after ICVinjection with 250 μg Cy3-labled hsiRNA variants (right panels). Imagestaken with Leica tiling array microscope at 10× and at identical laserintensity. Nuclei (blue); Cy3-hsiRNA (red). Chol-hsiRNA mainly stayedaround the injected ventricle with marginal distribution to the distalsides of the brain. DHA-hsiRNA shows better distribution. PC-DHA andDi-hsiRNAs shows most diffuse distribution with clear delivery tocortex, striatum, and even cerebellum. Scale bar=900 μm.

FIG. 93 depicts a synthetic protocol for PC-DHA-functionalized solidsupport.

FIG. 94 depicts a synthetic protocol for DI-functionalized solidsupport.

FIGS. 95A-95C depict di-hsiRNA discovery. (A) Chemical composition ofthe four bi-products from calciferol-hsiRNA synthesis (analytical HPLCof the crude synthesis). (B) Efficacy of bi-products in HeLa cells, 72hours, QuantiGene®. All compounds were equally active. (C) A single,unilateral intrastriatal injection (25 μg) of each Cy3-hsiRNAbi-product, 48 hours. Only di-hsiRNAs showed broad distribution withpreferential neuronal uptake.

FIG. 96 depicts an hsiANTIDOTE antisense oligonucleotide carrying highaffinity modification (LNA) designed to be fully complementary to thehsiRNA antisense strand seed region.

FIG. 97 depicts a cholesterol and endocytic peptide (proton sponge)conjugated hsiRNA. Fig. discloses SEQ ID NO: 1329.

FIG. 98A-98B depicts a solution-phase synthetic protocol for aGM1-conjugated hsiRNA.

FIG. 99 depicts chemical structures for DHA-conjugates (g1DHA) andPC-DHA hsiRNA conjugates (g2DHA).

FIG. 100 depicts a solid-phase synthetic protocol for PC-DHA hsiRNAconjugates.

FIG. 101 depicts a solution-phase synthetic protocol for PC-DHA hsiRNAconjugates.

FIGS. 102A-102D depict that full metabolic stabilization was essentialfor conjugate mediated siRNA delivery and duration of effect in vivo.(A, B) Compared to hsiRNA (A), FM-hsiRNA (B) showed significantlyenhanced distribution and retention in tissues after intravenous (IV)and CSF (ICV) administration. Wild-type pregnant mice (E15) wereinjected with 10 mg/kg IV or 60 μg, ICV. Tissues were imaged at 10× on aLeica tiling fluorescent microscope at identical laser intensity.HsiRNAs (red); nuclei (blue). Scale bar=900 μm. (C) Intact guide strandin tissues quantified 5 days after IV injection (n=3, mean±SEM). (D)FM-hsiRNAs silence Htt mRNA in mouse striatum one month after injection(12 μg, intrastriatal). Partially modified hsiRNAs silence for less thantwo weeks.

FIGS. 103A-103C depict PC-DHA-hsiRNAs efficacy and safety in mouse brainin vivo. (A) Htt mRNA levels in striatum and cortex 1 week afterinjecting 25 or 50 μg DHA-hsiRNA. ***P<0.0001 relative to both aCSF andNTC. (B) No detectable innate immune activation occurred at dose levels20-fold higher than the effective dose (data shown for total microgliafor DHA-hsiRNA). (C) Normal neuronal viability based on DARP32 levels.Note the toxic dose (red bar) for chol-hsiRNA.

FIGS. 104A-104C show that di-hsiRNA exhibited wide distribution andefficacy in mouse brain. (A) Robust and uniform distribution ofCy3-Di-hsiRNA throughout the brain, visually and histologically, withclear neuronal uptake 48 hours after ICV injection (250 μg, CSF, bothsides), scale bar=100 μm. (B) Htt mRNA silencing in cortex and striatum7 days after single intrastriatal injection (25 μg). (C) hsiRNAaccumulation in tissues 7 days after injection (PNA assay).

FIGS. 105A-105B show that di-hsiRNAs exhibited wide distribution andefficacy in the mouse spinal cord after a bolus lumbar intrathecalinjection. (A) Chol-hsiRNAs showed a steep gradient of diffusion fromoutside to inside of spinal cord, but Di-hsiRNAs distribute widelythroughout the spinal cord. Animals were injected intrathecally with 75μg Cy3-Chol-hsiRNA or Cy3-Di-hsiRNA. Scale bar=100 μm. (B) Robust HttmRNA silencing was observed in all regions of spinal cord (7 dayspost-injection, n=6).

FIG. 106 depicts a PNA (Peptide Nucleic Acid)-based assay for detectionof hsiRNA guide strand in mouse tissues. Tissues were lysed, debrisseparated by precipitation, and the PNA-guide strand duplex purified byHPLC (DNAPac P100, 50% water 50% acetonitrile, salt gradient 0-1MNaClO₄).

FIG. 107 depicts targeting of the kidney by PC-DHA-hsiRNA.

FIG. 108 depicts GM1-hsiRNA internalization and GM1-hsiRNA-mediated httmRNA silencing.

FIG. 109 depicts GM1-hsiRNA brain distribution.

FIGS. 110A-110G show that systemically-administered fully modified (FM)hsiRNA exhibits dramatically enhanced tissue distribution and efficacyin vivo. (A) Tissue distribution of Cy3-hsiRNA and Cy3-FM-hsiRNAsFLT1(red) 10 mg/kg IV injection. Nuclei stained with DAPI (blue). All imageswere acquired at identical settings. (B-E) Guide strand quantificationby PNA hybridization-based assay (B) 10 mg/kg, IV, 24 hours (C) 10mg/kg, SC, 24 hours (D) 2×20 mg/kg, IV, 120 hours, (n=7) (E) 2×15 mg/kg,IV, 120 hours, (n=12). (F, G) Quantification of sFLT1 mRNA silencingafter (F) 2×20 mg/kg, C57B6 mice, (n=3, PBS; n=7, FM-hsiRNAsFLT), (G)2×15 mg/kg, CD1 mice. (n=12, for PBS; n=6, NTC; n=12, FM-hsiRNAsFLT1).mRNA levels were measured 120 hours after injection with QuantiGene®(Affymetrix) assay, normalized to housekeeping gene FLT1, and presentedas percent of PBS treated control. All error bars represent mean±SD.***, P<0.001; ****, P<0.0001.

FIGS. 111A-111G show that fully modified hsiRNAs are broadly distributedthroughout the brain and demonstrate higher potency and longer durationof silencing upon local administration. hsiRNAHTT (FIG. 111A) andFM-hsiRNAHTT (FIG. 111B, FIG. 111C, FIG. 111D, FIG. 111E) were injectedICV, distribution through the sagittal section of the brain after 48hours is shown. Nuclei stained with DAPI (blue). Cy3-hsiRNA (red). (FIG.111F, FIG. 111G) hsiRNAHTT and FM-hsiRNAHTT were unilaterally injectedinto the striatum and level of HTT mRNA was measured using QuantiGene®(Affymetrix) after (FIG. 111F) 5 days or (FIG. 111G) 7, 14 and 28 days,normalized to housekeeping gene, PPIB, and presented as percent ofuntreated control (n=8 mice, mean±SD). NTC=non-targeting control;CSF=artificial cerebrospinal fluid All error bars represent mean±SD. **,P<0.01; ***, P<0.001; ****, P<0.0001.

DETAILED DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS

Novel huntingtin target sequences are provided. Also provided are novelsiRNAs that target the novel huntingtin target sequences of theinvention.

Generally, nomenclature used in connection with cell and tissue culture,molecular biology, immunology, microbiology, genetics and protein andnucleic acid chemistry and hybridization described herein are thosewell-known and commonly used in the art. The methods and techniquesprovided herein are generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification unless otherwise indicated. Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications, as commonly accomplished in the art or as describedherein. The nomenclature used in connection with, and the laboratoryprocedures and techniques of, analytical chemistry, synthetic organicchemistry, and medicinal and pharmaceutical chemistry described hereinare those well-known and commonly used in the art. Standard techniquesare used for chemical syntheses, chemical analyses, pharmaceuticalpreparation, formulation, and delivery, and treatment of patients.

Unless otherwise defined herein, scientific and technical terms usedherein have the meanings that are commonly understood by those ofordinary skill in the art. In the event of any latent ambiguity,definitions provided herein take precedent over any dictionary orextrinsic definition. Unless otherwise required by context, singularterms shall include pluralities and plural terms shall include thesingular. The use of “or” means “and/or” unless stated otherwise. Theuse of the term “including,” as well as other forms, such as “includes”and “included,” is not limiting.

So that the invention may be more readily understood, certain terms arefirst defined.

The term “nucleoside” refers to a molecule having a purine or pyrimidinebase covalently linked to a ribose or deoxyribose sugar. Exemplarynucleosides include adenosine, guanosine, cytidine, uridine andthymidine. Additional exemplary nucleosides include inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine,2N-methylguanosine and 2,2N,N-dimethylguanosine (also referred to as“rare” nucleosides). The term “nucleotide” refers to a nucleoside havingone or more phosphate groups joined in ester linkages to the sugarmoiety. Exemplary nucleotides include nucleoside monophosphates,diphosphates and triphosphates. The terms “polynucleotide” and “nucleicacid molecule” are used interchangeably herein and refer to a polymer ofnucleotides joined together by a phosphodiester or phosphorothioatelinkage between 5′ and 3′ carbon atoms.

The term “RNA” or “RNA molecule” or “ribonucleic acid molecule” refersto a polymer of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30,or more ribonucleotides). The term “DNA” or “DNA molecule” ordeoxyribonucleic acid molecule” refers to a polymer ofdeoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., byDNA replication or transcription of DNA, respectively). RNA can bepost-transcriptionally modified. DNA and RNA can also be chemicallysynthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA,respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA anddsDNA, respectively). “mRNA” or “messenger RNA” is single-stranded RNAthat specifies the amino acid sequence of one or more polypeptidechains. This information is translated during protein synthesis whenribosomes bind to the mRNA.

As used herein, the term “small interfering RNA” (“siRNA”) (alsoreferred to in the art as “short interfering RNAs”) refers to an RNA (orRNA analog) comprising between about 10-50 nucleotides (or nucleotideanalogs) which is capable of directing or mediating RNA interference.Preferably, a siRNA comprises between about 15-30 nucleotides ornucleotide analogs, more preferably between about 16-25 nucleotides (ornucleotide analogs), even more preferably between about 18-23nucleotides (or nucleotide analogs), and even more preferably betweenabout 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21 or 22nucleotides or nucleotide analogs). The term “short” siRNA refers to asiRNA comprising about 21 nucleotides (or nucleotide analogs), forexample, 19, 20, 21 or 22 nucleotides. The term “long” siRNA refers to asiRNA comprising about 24-25 nucleotides, for example, 23, 24, 25 or 26nucleotides. Short siRNAs may, in some instances, include fewer than 19nucleotides, e.g., 16, 17 or 18 nucleotides, provided that the shortersiRNA retains the ability to mediate RNAi. Likewise, long siRNAs may, insome instances, include more than 26 nucleotides, provided that thelonger siRNA retains the ability to mediate RNAi absent furtherprocessing, e.g., enzymatic processing, to a short siRNA.

The term “nucleotide analog” or “altered nucleotide” or “modifiednucleotide” refers to a non-standard nucleotide, including non-naturallyoccurring ribonucleotides or deoxyribonucleotides. Exemplary nucleotideanalogs are modified at any position so as to alter certain chemicalproperties of the nucleotide yet retain the ability of the nucleotideanalog to perform its intended function. Examples of positions of thenucleotide which may be derivatized include the 5 position, e.g.,5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine,5-propenyl uridine, etc.; the 6 position, e.g., 6-(2-amino)propyluridine; the 8-position for adenosine and/or guanosines, e.g., 8-bromoguanosine, 8-chloro guanosine, 8-fluoroguanosine, etc. Nucleotideanalogs also include deaza nucleotides, e.g., 7-deaza-adenosine; O- andN-modified (e.g., alkylated, e.g., N6-methyl adenosine, or as otherwiseknown in the art) nucleotides; and other heterocyclically modifiednucleotide analogs such as those described in Herdewijn, AntisenseNucleic Acid Drug Dev., 2000 Aug. 10(4):297-310.

Nucleotide analogs may also comprise modifications to the sugar portionof the nucleotides. For example the 2′ OH-group may be replaced by agroup selected from H, OR, R, F, Cl, Br, I, SH, SR, NH₂, NHR, NR₂, COOR,or OR, wherein R is substituted or unsubstituted C₁-C₆ alkyl, alkenyl,alkynyl, aryl, etc. Other possible modifications include those describedin U.S. Pat. Nos. 5,858,988, and 6,291,438.

The phosphate group of the nucleotide may also be modified, e.g., bysubstituting one or more of the oxygens of the phosphate group withsulfur (e.g., phosphorothioates), or by making other substitutions whichallow the nucleotide to perform its intended function such as describedin, for example, Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr.10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct.11(5): 317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001Apr. 11(2):77-85, and U.S. Pat. No. 5,684,143. Certain of theabove-referenced modifications (e.g., phosphate group modifications)preferably decrease the rate of hydrolysis of, for example,polynucleotides comprising said analogs in vivo or in vitro.

The term “oligonucleotide” refers to a short polymer of nucleotidesand/or nucleotide analogs. The term “RNA analog” refers to anpolynucleotide (e.g., a chemically synthesized polynucleotide) having atleast one altered or modified nucleotide as compared to a correspondingunaltered or unmodified RNA but retaining the same or similar nature orfunction as the corresponding unaltered or unmodified RNA. As discussedabove, the oligonucleotides may be linked with linkages which result ina lower rate of hydrolysis of the RNA analog as compared to an RNAmolecule with phosphodiester linkages. For example, the nucleotides ofthe analog may comprise methylenediol, ethylene diol, oxymethylthio,oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phosphoroamidate,and/or phosphorothioate linkages. Preferred RNA analogues include sugar-and/or backbone-modified ribonucleotides and/or deoxyribonucleotides.Such alterations or modifications can further include addition ofnon-nucleotide material, such as to the end(s) of the RNA or internally(at one or more nucleotides of the RNA). An RNA analog need only besufficiently similar to natural RNA that it has the ability to mediate(mediates) RNA interference.

As used herein, the term “RNA interference” (“RNAi”) refers to aselective intracellular degradation of RNA. RNAi occurs in cellsnaturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAiproceeds via fragments cleaved from free dsRNA which direct thedegradative mechanism to other similar RNA sequences. Alternatively,RNAi can be initiated by the hand of man, for example, to silence theexpression of target genes.

An RNAi agent, e.g., an RNA silencing agent, having a strand which is“sequence sufficiently complementary to a target mRNA sequence to directtarget-specific RNA interference (RNAi)” means that the strand has asequence sufficient to trigger the destruction of the target mRNA by theRNAi machinery or process.

As used herein, the term “isolated RNA” (e.g., “isolated siRNA” or“isolated siRNA precursor”) refers to RNA molecules which aresubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized.

As used herein, the term “RNA silencing” refers to a group ofsequence-specific regulatory mechanisms (e.g. RNA interference (RNAi),transcriptional gene silencing (TGS), post-transcriptional genesilencing (PTGS), quelling, co-suppression, and translationalrepression) mediated by RNA molecules which result in the inhibition or“silencing” of the expression of a corresponding protein-coding gene.RNA silencing has been observed in many types of organisms, includingplants, animals, and fungi.

The term “discriminatory RNA silencing” refers to the ability of an RNAmolecule to substantially inhibit the expression of a “first” or“target” polynucleotide sequence while not substantially inhibiting theexpression of a “second” or “non-target” polynucleotide sequence,” e.g.,when both polynucleotide sequences are present in the same cell. Incertain embodiments, the target polynucleotide sequence corresponds to atarget gene, while the non-target polynucleotide sequence corresponds toa non-target gene. In other embodiments, the target polynucleotidesequence corresponds to a target allele, while the non-targetpolynucleotide sequence corresponds to a non-target allele. In certainembodiments, the target polynucleotide sequence is the DNA sequenceencoding the regulatory region (e.g. promoter or enhancer elements) of atarget gene. In other embodiments, the target polynucleotide sequence isa target mRNA encoded by a target gene.

The term “in vitro” has its art recognized meaning, e.g., involvingpurified reagents or extracts, e.g., cell extracts. The term “in vivo”also has its art recognized meaning, e.g., involving living cells, e.g.,immortalized cells, primary cells, cell lines, and/or cells in anorganism.

As used herein, the term “transgene” refers to any nucleic acidmolecule, which is inserted by artifice into a cell, and becomes part ofthe genome of the organism that develops from the cell. Such a transgenemay include a gene that is partly or entirely heterologous (i.e.,foreign) to the transgenic organism, or may represent a gene homologousto an endogenous gene of the organism. The term “transgene” also means anucleic acid molecule that includes one or more selected nucleic acidsequences, e.g., DNAs, that encode one or more engineered RNAprecursors, to be expressed in a transgenic organism, e.g., animal,which is partly or entirely heterologous, i.e., foreign, to thetransgenic animal, or homologous to an endogenous gene of the transgenicanimal, but which is designed to be inserted into the animal's genome ata location which differs from that of the natural gene. A transgeneincludes one or more promoters and any other DNA, such as introns,necessary for expression of the selected nucleic acid sequence, alloperably linked to the selected sequence, and may include an enhancersequence.

A gene “involved” in a disease or disorder includes a gene, the normalor aberrant expression or function of which effects or causes thedisease or disorder or at least one symptom of said disease or disorder.

The term “gain-of-function mutation” as used herein, refers to anymutation in a gene in which the protein encoded by said gene (i.e., themutant protein) acquires a function not normally associated with theprotein (i.e., the wild type protein) causes or contributes to a diseaseor disorder. The gain-of-function mutation can be a deletion, addition,or substitution of a nucleotide or nucleotides in the gene which givesrise to the change in the function of the encoded protein. In oneembodiment, the gain-of-function mutation changes the function of themutant protein or causes interactions with other proteins. In anotherembodiment, the gain-of-function mutation causes a decrease in orremoval of normal wild-type protein, for example, by interaction of thealtered, mutant protein with said normal, wild-type protein.

As used herein, the term “target gene” is a gene whose expression is tobe substantially inhibited or “silenced.” This silencing can be achievedby RNA silencing, e.g., by cleaving the mRNA of the target gene ortranslational repression of the target gene. The term “non-target gene”is a gene whose expression is not to be substantially silenced. In oneembodiment, the polynucleotide sequences of the target and non-targetgene (e.g. mRNA encoded by the target and non-target genes) can differby one or more nucleotides. In another embodiment, the target andnon-target genes can differ by one or more polymorphisms (e.g., SingleNucleotide Polymorphisms or SNPs). In another embodiment, the target andnon-target genes can share less than 100% sequence identity. In anotherembodiment, the non-target gene may be a homologue (e.g. an orthologueor paralogue) of the target gene.

A “target allele” is an allele (e.g., a SNP allele) whose expression isto be selectively inhibited or “silenced.” This silencing can beachieved by RNA silencing, e.g., by cleaving the mRNA of the target geneor target allele by a siRNA. The term “non-target allele” is a allelewhose expression is not to be substantially silenced. In certainembodiments, the target and non-target alleles can correspond to thesame target gene. In other embodiments, the target allele correspondsto, or is associated with, a target gene, and the non-target allelecorresponds to, or is associated with, a non-target gene. In oneembodiment, the polynucleotide sequences of the target and non-targetalleles can differ by one or more nucleotides. In another embodiment,the target and non-target alleles can differ by one or more allelicpolymorphisms (e.g., one or more SNPs). In another embodiment, thetarget and non-target alleles can share less than 100% sequenceidentity.

The term “polymorphism” as used herein, refers to a variation (e.g., oneor more deletions, insertions, or substitutions) in a gene sequence thatis identified or detected when the same gene sequence from differentsources or subjects (but from the same organism) are compared. Forexample, a polymorphism can be identified when the same gene sequencefrom different subjects are compared. Identification of suchpolymorphisms is routine in the art, the methodologies being similar tothose used to detect, for example, breast cancer point mutations.Identification can be made, for example, from DNA extracted from asubject's lymphocytes, followed by amplification of polymorphic regionsusing specific primers to said polymorphic region. Alternatively, thepolymorphism can be identified when two alleles of the same gene arecompared. In particular embodiments, the polymorphism is a singlenucleotide polymorphism (SNP).

A variation in sequence between two alleles of the same gene within anorganism is referred to herein as an “allelic polymorphism.” In certainembodiments, the allelic polymorphism corresponds to a SNP allele. Forexample, the allelic polymorphism may comprise a single nucleotidevariation between the two alleles of a SNP. The polymorphism can be at anucleotide within a coding region but, due to the degeneracy of thegenetic code, no change in amino acid sequence is encoded.Alternatively, polymorphic sequences can encode a different amino acidat a particular position, but the change in the amino acid does notaffect protein function. Polymorphic regions can also be found innon-encoding regions of the gene. In exemplary embodiments, thepolymorphism is found in a coding region of the gene or in anuntranslated region (e.g., a 5′ UTR or 3′ UTR) of the gene.

As used herein, the term “allelic frequency” is a measure (e.g.,proportion or percentage) of the relative frequency of an allele (e.g.,a SNP allele) at a single locus in a population of individuals. Forexample, where a population of individuals carry n loci of a particularchromosomal locus (and the gene occupying the locus) in each of theirsomatic cells, then the allelic frequency of an allele is the fractionor percentage of loci that the allele occupies within the population. Inparticular embodiments, the allelic frequency of an allele (e.g., an SNPallele) is at least 10% (e.g., at least 15%, 20%, 25%, 30%, 35%, 40% ormore) in a sample population.

As used herein, the term “sample population” refers to a population ofindividuals comprising a statistically significant number ofindividuals. For example, the sample population may comprise 50, 75,100, 200, 500, 1000 or more individuals. In particular embodiments, thesample population may comprise individuals which share at least oncommon disease phenotype (e.g., a gain-of-function disorder) or mutation(e.g., a gain-of-function mutation).

As used herein, the term “heterozygosity” refers to the fraction ofindividuals within a population that are heterozygous (e.g., contain twoor more different alleles) at a particular locus (e.g., at a SNP).Heterozygosity may be calculated for a sample population using methodsthat are well known to those skilled in the art.

The term “polyglutamine domain,” as used herein, refers to a segment ordomain of a protein that consist of a consecutive glutamine residueslinked to peptide bonds. In one embodiment the consecutive regionincludes at least 5 glutamine residues.

The term “expanded polyglutamine domain” or “expanded polyglutaminesegment,” as used herein, refers to a segment or domain of a proteinthat includes at least 35 consecutive glutamine residues linked bypeptide bonds. Such expanded segments are found in subjects afflictedwith a polyglutamine disorder, as described herein, whether or not thesubject has shown to manifest symptoms.

The term “trinucleotide repeat” or “trinucleotide repeat region” as usedherein, refers to a segment of a nucleic acid sequence e.g.,) thatconsists of consecutive repeats of a particular trinucleotide sequence.In one embodiment, the trinucleotide repeat includes at least 5consecutive trinucleotide sequences. Exemplary trinucleotide sequencesinclude, but are not limited to, CAG, CGG, GCC, GAA, CTG and/or CGG.

The term “trinucleotide repeat diseases” as used herein, refers to anydisease or disorder characterized by an expanded trinucleotide repeatregion located within a gene, the expanded trinucleotide repeat regionbeing causative of the disease or disorder. Examples of trinucleotiderepeat diseases include, but are not limited to spino-cerebellar ataxiatype 12 spino-cerebellar ataxia type 8, fragile X syndrome, fragile XEmental retardation, Friedreich's ataxia and myotonic dystrophy.Exemplary trinucleotide repeat diseases for treatment according to thepresent invention are those characterized or caused by an expandedtrinucleotide repeat region at the 5′ end of the coding region of agene, the gene encoding a mutant protein which causes or is causative ofthe disease or disorder. Certain trinucleotide diseases, for example,fragile X syndrome, where the mutation is not associated with a codingregion may not be suitable for treatment according to the methodologiesof the present invention, as there is no suitable mRNA to be targeted byRNAi. By contrast, disease such as Friedreich's ataxia may be suitablefor treatment according to the methodologies of the invention because,although the causative mutation is not within a coding region (i.e.,lies within an intron), the mutation may be within, for example, an mRNAprecursor (e.g., a pre-spliced mRNA precursor).

The term “polyglutamine disorder” as used herein, refers to any diseaseor disorder characterized by an expanded of a (CAG)n repeats at the 5′end of the coding region (thus encoding an expanded polyglutamine regionin the encoded protein). In one embodiment, polyglutamine disorders arecharacterized by a progressive degeneration of nerve cells. Examples ofpolyglutamine disorders include but are not limited to: Huntington'sdisease, spino-cerebellar ataxia type 1, spino-cerebellar ataxia type 2,spino-cerebellar ataxia type 3 (also known as Machado-Joseph disease),and spino-cerebellar ataxia type 6, spino-cerebellar ataxia type 7 anddentatoiubral-pallidoluysian atrophy.

The phrase “examining the function of a gene in a cell or organism”refers to examining or studying the expression, activity, function orphenotype arising therefrom.

As used herein, the term “RNA silencing agent” refers to an RNA which iscapable of inhibiting or “silencing” the expression of a target gene. Incertain embodiments, the RNA silencing agent is capable of preventingcomplete processing (e.g., the full translation and/or expression) of amRNA molecule through a post-transcriptional silencing mechanism. RNAsilencing agents include small (<50 b.p.), noncoding RNA molecules, forexample RNA duplexes comprising paired strands, as well as precursorRNAs from which such small non-coding RNAs can be generated. ExemplaryRNA silencing agents include siRNAs, miRNAs, siRNA-like duplexes,antisense oligonucleotides, GAPMER molecules, and dual-functionoligonucleotides as well as precursors thereof. In one embodiment, theRNA silencing agent is capable of inducing RNA interference. In anotherembodiment, the RNA silencing agent is capable of mediatingtranslational repression.

As used herein, the term “rare nucleotide” refers to a naturallyoccurring nucleotide that occurs infrequently, including naturallyoccurring deoxyribonucleotides or ribonucleotides that occurinfrequently, e.g., a naturally occurring ribonucleotide that is notguanosine, adenosine, cytosine, or uridine. Examples of rare nucleotidesinclude, but are not limited to, inosine, 1-methyl inosine,pseudouridine, 5,6-dihydrouridine, ribothymidine, ²N-methylguanosine and^(2,2)N,N-dimethylguanosine.

The term “engineered,” as in an engineered RNA precursor, or anengineered nucleic acid molecule, indicates that the precursor ormolecule is not found in nature, in that all or a portion of the nucleicacid sequence of the precursor or molecule is created or selected by ahuman. Once created or selected, the sequence can be replicated,translated, transcribed, or otherwise processed by mechanisms within acell. Thus, an RNA precursor produced within a cell from a transgenethat includes an engineered nucleic acid molecule is an engineered RNAprecursor.

As used herein, the term “microRNA” (“miRNA”), also referred to in theart as “small temporal RNAs” (“stRNAs”), refers to a small (10-50nucleotide) RNA which are genetically encoded (e.g., by viral,mammalian, or plant genomes) and are capable of directing or mediatingRNA silencing. An “miRNA disorder” shall refer to a disease or disordercharacterized by an aberrant expression or activity of an miRNA.

As used herein, the term “dual functional oligonucleotide” refers to aRNA silencing agent having the formula T-L-μ, wherein T is an mRNAtargeting moiety, L is a linking moiety, and μ is a miRNA recruitingmoiety. As used herein, the terms “mRNA targeting moiety,” “targetingmoiety,” “mRNA targeting portion” or “targeting portion” refer to adomain, portion or region of the dual functional oligonucleotide havingsufficient size and sufficient complementarity to a portion or region ofan mRNA chosen or targeted for silencing (i.e., the moiety has asequence sufficient to capture the target mRNA). As used herein, theterm “linking moiety” or “linking portion” refers to a domain, portionor region of the RNA-silencing agent which covalently joins or links themRNA.

As used herein, the term “antisense strand” of an RNA silencing agent,e.g., an siRNA or RNA silencing agent, refers to a strand that issubstantially complementary to a section of about 10-50 nucleotides,e.g., about 15-30, 16-25, 18-23 or 19-22 nucleotides of the mRNA of thegene targeted for silencing. The antisense strand or first strand hassequence sufficiently complementary to the desired target mRNA sequenceto direct target-specific silencing, e.g., complementarity sufficient totrigger the destruction of the desired target mRNA by the RNAi machineryor process (RNAi interference) or complementarity sufficient to triggertranslational repression of the desired target mRNA.

The term “sense strand” or “second strand” of an RNA silencing agent,e.g., an siRNA or RNA silencing agent, refers to a strand that iscomplementary to the antisense strand or first strand. Antisense andsense strands can also be referred to as first or second strands, thefirst or second strand having complementarity to the target sequence andthe respective second or first strand having complementarity to saidfirst or second strand. miRNA duplex intermediates or siRNA-likeduplexes include a miRNA strand having sufficient complementarity to asection of about 10-50 nucleotides of the mRNA of the gene targeted forsilencing and a miRNA* strand having sufficient complementarity to forma duplex with the miRNA strand.

As used herein, the term “guide strand” refers to a strand of an RNAsilencing agent, e.g., an antisense strand of an siRNA duplex or siRNAsequence, that enters into the RISC complex and directs cleavage of thetarget mRNA.

As used herein, the term “asymmetry,” as in the asymmetry of the duplexregion of an RNA silencing agent (e.g., the stem of an shRNA), refers toan inequality of bond strength or base pairing strength between thetermini of the RNA silencing agent (e.g., between terminal nucleotideson a first strand or stem portion and terminal nucleotides on anopposing second strand or stem portion), such that the 5′ end of onestrand of the duplex is more frequently in a transient unpaired, e.g.,single-stranded, state than the 5′ end of the complementary strand. Thisstructural difference determines that one strand of the duplex ispreferentially incorporated into a RISC complex. The strand whose 5′ endis less tightly paired to the complementary strand will preferentiallybe incorporated into RISC and mediate RNAi.

As used herein, the term “bond strength” or “base pair strength” refersto the strength of the interaction between pairs of nucleotides (ornucleotide analogs) on opposing strands of an oligonucleotide duplex(e.g., an siRNA duplex), due primarily to H-bonding, van der Waalsinteractions, and the like between said nucleotides (or nucleotideanalogs).

As used herein, the “5′ end,” as in the 5′ end of an antisense strand,refers to the 5′ terminal nucleotides, e.g., between one and about 5nucleotides at the 5′ terminus of the antisense strand. As used herein,the “3′ end,” as in the 3′ end of a sense strand, refers to the region,e.g., a region of between one and about 5 nucleotides, that iscomplementary to the nucleotides of the 5′ end of the complementaryantisense strand.

As used herein the term “destabilizing nucleotide” refers to a firstnucleotide or nucleotide analog capable of forming a base pair withsecond nucleotide or nucleotide analog such that the base pair is oflower bond strength than a conventional base pair (i.e., Watson-Crickbase pair). In certain embodiments, the destabilizing nucleotide iscapable of forming a mismatch base pair with the second nucleotide. Inother embodiments, the destabilizing nucleotide is capable of forming awobble base pair with the second nucleotide. In yet other embodiments,the destabilizing nucleotide is capable of forming an ambiguous basepair with the second nucleotide.

As used herein, the term “base pair” refers to the interaction betweenpairs of nucleotides (or nucleotide analogs) on opposing strands of anoligonucleotide duplex (e.g., a duplex formed by a strand of a RNAsilencing agent and a target mRNA sequence), due primarily to H-bonding,van der Waals interactions, and the like between said nucleotides (ornucleotide analogs). As used herein, the term “bond strength” or “basepair strength” refers to the strength of the base pair.

As used herein, the term “mismatched base pair” refers to a base pairconsisting of non-complementary or non-Watson-Crick base pairs, forexample, not normal complementary G:C, A:T or A:U base pairs. As usedherein the term “ambiguous base pair” (also known as anon-discriminatory base pair) refers to a base pair formed by auniversal nucleotide.

As used herein, term “universal nucleotide” (also known as a “neutralnucleotide”) include those nucleotides (e.g. certain destabilizingnucleotides) having a base (a “universal base” or “neutral base”) thatdoes not significantly discriminate between bases on a complementarypolynucleotide when forming a base pair. Universal nucleotides arepredominantly hydrophobic molecules that can pack efficiently intoantiparallel duplex nucleic acids (e.g., double-stranded DNA or RNA) dueto stacking interactions. The base portion of universal nucleotidestypically comprise a nitrogen-containing aromatic heterocyclic moiety.

As used herein, the terms “sufficient complementarity” or “sufficientdegree of complementarity” mean that the RNA silencing agent has asequence (e.g. in the antisense strand, mRNA targeting moiety or miRNArecruiting moiety) which is sufficient to bind the desired target RNA,respectively, and to trigger the RNA silencing of the target mRNA.

As used herein, the term “translational repression” refers to aselective inhibition of mRNA translation. Natural translationalrepression proceeds via miRNAs cleaved from shRNA precursors. Both RNAiand translational repression are mediated by RISC. Both RNAi andtranslational repression occur naturally or can be initiated by the handof man, for example, to silence the expression of target genes.

Various methodologies of the instant invention include step thatinvolves comparing a value, level, feature, characteristic, property,etc. to a “suitable control,” referred to interchangeably herein as an“appropriate control.” A “suitable control” or “appropriate control” isany control or standard familiar to one of ordinary skill in the artuseful for comparison purposes. In one embodiment, a “suitable control”or “appropriate control” is a value, level, feature, characteristic,property, etc. determined prior to performing an RNAi methodology, asdescribed herein. For example, a transcription rate, mRNA level,translation rate, protein level, biological activity, cellularcharacteristic or property, genotype, phenotype, etc. can be determinedprior to introducing an RNA silencing agent of the invention into a cellor organism. In another embodiment, a “suitable control” or “appropriatecontrol” is a value, level, feature, characteristic, property, etc.determined in a cell or organism, e.g., a control or normal cell ororganism, exhibiting, for example, normal traits. In yet anotherembodiment, a “suitable control” or “appropriate control” is apredefined value, level, feature, characteristic, property, etc.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and example are illustrative only and not intendedto be limiting.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Polyglutamine Disorders

Polyglutamine disorders are a class of disease or disorderscharacterized by a common genetic mutation. In particular, the diseaseor disorders are characterized by an expanded repeat of thetrinucleotide CAG which gives rise, in the encoded protein, to anexpanded stretch of glutamine residues. Polyglutamine disorders aresimilar in that the diseases are characterized by a progressivedegeneration of nerve cells. Despite their similarities, polyglutaminedisorders occur on different chromosomes and thus occur on entirelydifferent segments of DNA. Examples of polyglutamine disorders includeHuntington's disease, Dentatorubropallidoluysian Atrophy, SpinobulbarMuscular atrophy, Spinocerebellar Ataxia Type 1, Spinocerebellar AtaxiaType 2, Spinocerebellar Ataxia Type 3, Spinocerebellar Ataxia Type 6 andSpinocerebellar Ataxia Type 7.

Polyglutamine disorders of the invention are characterized by, e.g.,domains having between about 30 to 35 glutamine residues, between about35 to 40 glutamine residues, between about 40 to 45 glutamine residuesor having about 45 or more glutamine residues. The polyglutamine domaintypically contains consecutive glutamine residues (Q n>36).

II. Huntington Disease

In some embodiments, the RNA silencing agents of the invention aredesigned to target polymorphisms (e.g. single nucleotide polymorphisms)in the mutant human huntingtin protein (htt) for the treatment ofHuntington's disease.

Huntington's disease, inherited as an autosomal dominant disease, causesimpaired cognition and motor disease. Patients can live more than adecade with severe debilitation, before premature death from starvationor infection. The disease begins in the fourth or fifth decade for mostcases, but a subset of patients manifest disease in teenage years. Thegenetic mutation for Huntington's disease is a lengthened CAG repeat inthe huntingtin gene. CAG repeats vary in number from 8 to 35 in normalindividuals (Kremer et al., 1994). The genetic mutation e.g., anincrease in length of the CAG repeats from normal (less than 36 in thehuntingtin gene to greater than 36 in the disease) is associated withthe synthesis of a mutant Huntingtin protein, which has greater than 36polyglutamates (Aronin et al., 1995). In general, individuals with 36 ormore CAG repeats will develop Huntington's disease. Prototypic for asmany as twenty other diseases with a lengthened CAG as the underlyingmutation, Huntington's disease still has no effective therapy. A varietyof interventions, such as interruption of apoptotic pathways, additionof reagents to boost mitochondrial efficiency, and blockade of NMDAreceptors, have shown promise in cell cultures and mouse model ofHuntington's disease. However, at best these approaches reveal a shortprolongation of cell or animal survival.

Huntington's disease complies with the central dogma of genetics: amutant gene serves as a template for production of a mutant mRNA; themutant mRNA then directs synthesis of a mutant protein (Aronin et al.,1995; DiFiglia et al., 1997). Without intending to be bound byscientific theory, it is thought that mutant huntingtin proteinaccumulates in selective neurons in the striatum and cortex, disrupts asyet determined cellular activities, and causes neuronal dysfunction anddeath (Aronin et al., 1999; Laforet et al., 2001). Because a single copyof a mutant gene suffices to cause Huntington's disease, the mostparsimonious treatment would render the mutant gene ineffective.Theoretical approaches might include stopping gene transcription ofmutant huntingtin, destroying mutant mRNA, and blocking translation.Each has the same outcome: loss of mutant huntingtin.

III. Huntingtin Gene

The disease gene linked to Huntington's disease is termed Huntingtin or(htt). The huntingtin locus is large, spanning 180 kb and consisting of67 exons. The huntingtin gene is widely expressed and is required fornormal development. It is expressed as 2 alternatively polyadenylatedforms displaying different relative abundance in various fetal and adulttissues. The larger transcript is approximately 13.7 kb and is expressedpredominantly in adult and fetal brain whereas the smaller transcript ofapproximately 10.3 kb is more widely expressed. The two transcriptsdiffer with respect to their 3′ untranslated regions (Lin et al., 1993).Both messages are predicted to encode a 348 kilodalton proteincontaining 3144 amino acids. The genetic defect leading to Huntington'sdisease is believed to confer a new property on the mRNA or alter thefunction of the protein.

The present invention targets huntingtin (e.g., wild-type and/or mutanthuntingtin) using RNA interference (Hutvagner et al., 2002). One strandof double-stranded RNA (siRNA) complements a target sequence within thehuntingtin mRNA. After introduction of siRNA into neurons, the siRNApartially unwinds, binds to polymorphic region within the huntingtinmRNA in a site-specific manner, and activates an mRNA nuclease. Thisnuclease cleaves the huntingtin mRNA, thereby halting translation of thehuntingtin (e.g., wild-type and/or mutant huntingtin). Cells ridthemselves of partially digested mRNA, thus precluding translation, orcells digest partially translated proteins. In certain embodiments,neurons survive on the wild-type huntingtin from the normal allele,preventing the ravages of mutant huntingtin by eliminating itsproduction.

In embodiments of the invention, RNA silencing agents of the inventionare capable of targeting one or more of the target sequences listed inFIG. 8. In certain exemplary embodiments, RNA silencing agents of theinvention are capable of targeting one or more of the target sequencesat one or more target sequences listed at gene positions selected fromthe group consisting of 1214, 1218, 1219, 1257, 1894, 1907, 2866, 4041,4049, 5301, 6016, 6579, 8603, 10125, 10146, 10150, 424, 456, 522, 527,878, 879, 908, 1024, 1165, 1207, 1212, 1217, 1220, 1223, 1227, 1229,1260, 1403, 1470, 1901, 1903, 2411, 2412, 2865, 3801, 4040, 4048, 4052,4055, 4083, 4275, 4372, 4374, 4376, 4425, 4562, 4692, 4721, 5200, 5443,5515, 8609, 10130, 10134, 10142, 10169, 10182, 10186, 10809, 11116,11129, 11134, 11147, 11412, 11426, 11443, 11659, 11666, 11677, 11863,11890, 11927, 11947, 12163, 12218, 12223, 12235, 12279, 12282, 12297,12309, 12313, 12331, 13136, 13398, 13403, 13423, 13428 of the human httgene (as set forth at FIG. 8). In certain exemplary embodiments, RNAsilencing agents of the invention are capable of targeting one or moreof the target sequences at one or more target sequences listed at genepositions selected from the group consisting of 5301, 10125, 10146,10150, 424, 878, 879, 4083, 4275, 4562, 4721, 5200, 10130, 10134, 10142,11116, 11129, 11134, 11147, 11412, 11426, 11443, 11659, 11666, 11677,11863, 11890, 11927, 11947, 12163, 12218, 12223, 12235, 12279, 12282,12297, 12331, 13136, 13423 and 13428 of the human htt gene (as set forthat FIG. 8). Particularly exemplary target sequences of the human httgene can be found at positions 10150 (5′ CAGUAAAGAGAUUAA 3′ (SEQ IDNO:1)), 10146 (5′ AUAUCAGUAAAGAGA 3′ (SEQ ID NO:2)) and 10125 (5′CUCAGGAUUUAAAAU 3′ (SEQ ID NO:3)). Genomic sequence for each targetsequence can be found in, for example, the publically available databasemaintained by the NCBI.

In certain exemplary embodiments, RNA silencing agents of the inventionthat are capable of targeting one or more of the target sequences at oneor more target sequences are set forth in Table 1, below, and in FIG. 21(which also includes exemplary modifications).

GCUGCCGGGA Accession Number Position Targeting region (20 mer)Targeting Region (30 mer) HTT NM_002111 .6 1214 GUCCAGGUUUAUGAACUGACAGCUUGUCCAGGUUUAUGAACUGACGUUAC HTT NM_002111 .6 1218AGGUUUAUGAACUGACGUUA UGUCCAGGUUUAUGAACUGACGUUACAUCA HTT NM_002111 .61219 GGUUUAUGAACUGACGUUAC GUCCAGGUUUAUGAACUGACGUUACAUCAU HTTNM_002111 .6 1257 ACCACAAUGUUGUGACCGGA CCAAGACCACAAUGUUGUGACCGGAGCCCUHTT NM_002111 .6 1894 UGUGUUAGACGGUACCGACAGAAAUUGUGUUAGACGGUACCGACAACCAG HTT NM_002111 .6 1907ACCGACAACCAGUAUUUGGG ACGGUACCGACAACCAGUAUUUGGGCCUGC HTT NM_002111 .62866 ACGAGUGCUCAAUAAUGUUG CAAGAACGAGUGCUCAAUAAUGUUGUCAUC HTTNM_002111 .6 4041 UGAAAUCCUGCUUUAGUCGA AUACCUGAAAUCCUGCUUUAGUCGAGAACCHTT NM_002111 .6 4049 UGCUUUAGUCGAGAACCAAUAAUCCUGCUUUAGUCGAGAACCAAUGAUGG HTT NM_002111 .6 5301GGGACAGUACUUCAACGCUA AGAUGGGGACAGUACUUCAACGCUAGAAGA HTT NM_002111 .66016 GGCAAUUCAGUCUCGUUGUG AUCCAGGCAAUUCAGUCUCGUUGUGAAAAC HTTNM_002111 .6 6579 GCCUGCUAGCUCCAUGCUUA CCUAAGCCUGCUAGCUCCAUGCUUAAGCCUHTT NM_002111 .6 8603 GCCCACUGCGUGAACAUUCAGGAUCGCCCACUGCGUGAACAUUCACAGCC HTT NM_002111 .6 10125UUCUUCUCAGGAUUUAAAAU CUCUUUUCUUCUCAGGAUUUAAAAUUUAAU HTT NM_002111 .610146 UAAUUAUAUCAGUAAAGAGA AAAUUUAAUUAUAUCAGUAAAGAGAUUAAU HTTNM_002111 .6 10150 UAUAUCAGUAAAGAGAUUAA UUAAUUAUAUCAGUAAAGAGAUUAAUUUUAHTT NM_002111 .6 424 ACUUUCAGCUACCAAGAAAG AAAGAACUUUCAGCUACCAAGAAAGACCGUHTT NM_002111 .6 456 AUUGUCUGACAAUAUGUGAA GAAUCAUUGUCUGACAAUAUGUGAAAACAUHTT NM_002111 .6 522 UUCUGGGCAUCGCUAUGGAA HTT NM_002111 .6 527GGCAUCGCUAUGGAACUUUU UUCUGGGCAUCGCUAUGGAACUUUUUCUGC HTT NM_002111 .6 878GCAAAUGACAAUGAAAUUAA AUUUUGCAAAUGACAAUGAAAUUAAGGUUU HTT NM_002111 .6 879CAAAUGACAAUGAAAUUAAG UUUUGCAAAUGACAAUGAAAUUAAGGUUUU HTT NM_002111 .6 908AAGGCCUUCAUAGCGAACCU UGUUAAAGGCCUUCAUAGCGAACCUGAAGU HTT NM_002111 .61024 ACUAAAUGUGCUCUUAGGCU UGGCUACUAAAUGUGCUCUUAGGCUUACUC HTTNM_002111 .6 1165 CGGAGUGACAAGGAAAGAAA AGCUUCGGAGUGACAAGGAAAGAAAUGGAAHTT NM_002111 .6 1207 GCAGCUUGUCCAGGUUUAUGGCAGAGCAGCUUGUCCAGGUUUAUGAACUG HTT NM_002111 .6 1212UUGUCCAGGUUUAUGAACUG GCAGCUUGUCCAGGUUUAUGAACUGACGUU HTT NM_002111 .61217 CAGGUUUAUGAACUGACGUU UUGUCCAGGUUUAUGAACUGACGUUACAUC HTTNM_002111 .6 1220 GUUUAUGAACUGACGUUACA UCCAGGUUUAUGAACUGACGUUACAUCAUAHTT NM_002111 .6 1223 UAUGAACUGACGUUACAUCAAGGUUUAUGAACUGACGUUACAUCAUACAC HTT NM_002111 .6 1227AACUGACGUUACAUCAUACA UUAUGAACUGACGUUACAUCAUACACAGCA HTT NM_002111 .61229 CUGACGUUACAUCAUACACA AUGAACUGACGUUACAUCAUACACAGCACC HTTNM_002111 .6 1260 ACAAUGUUGUGACCGGAGCC AGACCACAAUGUUGUGACCGGAGCCCUGGAHTT NM_002111 .6 1403 GGGAGUAUUGUGGAACUUAUGUAGUGGGAGUAUUGUGGAACUUAUAGCUG HTT NM_002111 .6 1470AAGGCAAAGUGCUCUUAGGA ACAAAAAGGCAAAGUGCUCUUAGGAGAAGA HTT NM_002111 .61901 GACGGUACCGACAACCAGUA UGUUAGACGGUACCGACAACCAGUAUUUGG HTTNM_002111 .6 1903 CGGUACCGACAACCAGUAUU UUAGACGGUACCGACAACCAGUAUUUGGGCHTT NM_002111 .6 2411 UUGAACUACAUCGAUCAUGGACAUCUUGAACUACAUCGAUCAUGGAGACC HTT NM_002111 .6 2412UGAACUACAUCGAUCAUGGA CAUCUUGAACUACAUCGAUCAUGGAGACCC HTT NM_002111 .62865 AACGAGUGCUCAAUAAUGUU GCAAGAACGAGUGCUCAAUAAUGUUGUCAU HTTNM_002111 .6 3801 GUCCUGUUACAACAAGUAAA CUCAGGUCCUGUUACAACAAGUAAAUCCUCHTT NM_002111 .6 4040 CUGAAAUCCUGCUUUAGUCGGAUACCUGAAAUCCUGCUUUAGUCGAGAAC HTT NM_002111 .6 4048CUGCUUUAGUCGAGAACCAA AAAUCCUGCUUUAGUCGAGAACCAAUGAUG HTT NM_002111 .64052 UUUAGUCGAGAACCAAUGAU CCUGCUUUAGUCGAGAACCAAUGAUGGCAA HTTNM_002111 .6 4055 AGUCGAGAACCAAUGAUGGC GCUUUAGUCGAGAACCAAUGAUGGCAACUGHTT NM_002111 .6 4083 GUGUUCAACAAUUGUUGAAGUGUUUGUGUUCAACAAUUGUUGAAGACUCU HTT NM_002111 .6 4275UGAGGAACAUGGUGCAGGCG CAGCCUGAGGAACAUGGUGCAGGCGGAGCA HTT NM_002111 .64372 UGUCACAAAGAACCGUGCAG ACGAGUGUCACAAAGAACCGUGCAGAUAAG HTTNM_002111 .6 4374 UCACAAAGAACCGUGCAGAU GAGUGUCACAAAGAACCGUGCAGAUAAGAAHTT NM_002111 .6 4376 ACAAAGAACCGUGCAGAUAAGUGUCACAAAGAACCGUGCAGAUAAGAAUG HTT NM_002111 .6 4425UUGAACCUCUUGUUAUAAAA UUUGUUUGAACCUCUUGUUAUAAAAGCUUU HTT NM_002111 .64562 UUUAUUGGCUUUGUAUUGAA AGGUGUUUAUUGGCUUUGUAUUGAAACAGU HTTNM_002111 .6 4692 UCAUUGGAAUUCCUAAAAUC ACAGAUCAUUGGAAUUCCUAAAAUCAUUCAHTT NM_002111 .6 4721 UGUGAUGGCAUCAUGGCCAGAGCUCUGUGAUGGCAUCAUGGCCAGUGGAA HTT NM_002111 .6 5200GAUUUCCCAGUCAACUGAAG GUUCUGAUUUCCCAGUCAACUGAAGAUAUU HTT NM_002111 .65443 GAGUGAGCAGCAACAUACUU GAAAUGAGUGAGCAGCAACAUACUUUCUAU HTTNM_002111 .6 5515 GUCUGGAAUGUUCCGGAGAA UUCAAGUCUGGAAUGUUCCGGAGAAUCACAHTT NM_002111 .6 8609 UGCGUGAACAUUCACAGCCACCCACUGCGUGAACAUUCACAGCCAGCAGC HTT NM_002111 .6 10130CUCAGGAUUUAAAAUUUAAU UUCUUCUCAGGAUUUAAAAUUUAAUUAUAU HTT NM_002111 .610134 GGAUUUAAAAUUUAAUUAUA UCUCAGGAUUUAAAAUUUAAUUAUAUCAGU HTTNM_002111 .6 10142 AAUUUAAUUAUAUCAGUAAA UUUAAAAUUUAAUUAUAUCAGUAAAGAGAUHTT NM_002111 .6 10169 AUUUUAACGUAACUCUUUCUGAUUAAUUUUAACGUAACUCUUUCUAUGCC HTT NM_002111 .6 10182UCUUUCUAUGCCCGUGUAAA GUAACUCUUUCUAUGCCCGUGUAAAGUAUG HTT NM_002111 .610186 UCUAUGCCCGUGUAAAGUAU CUCUUUCUAUGCCCGUGUAAAGUAUGUGAA HTTNM_002111 .6 10809 CUUUUAGUCAGGAGAGUGCA GACCCCUUUUAGUCAGGAGAGUGCAGAUCUHTT NM_002111 .6 11116 UGUUUUGGGUAUUGAAUGUGGUCGAUGUUUUGGGUAUUGAAUGUGGUAAG HTT NM_002111 .6 11129GAAUGUGGUAAGUGGAGGAA GUAUUGAAUGUGGUAAGUGGAGGAAAUGUU HTT NM_002111 .611134 UGGUAAGUGGAGGAAAUGUU GAAUGUGGUAAGUGGAGGAAAUGUUGGAAC HTTNM_002111 .6 11147 AAAUGUUGGAACUCUGUGCA GGAGGAAAUGUUGGAACUCUGUGCAGGUGCHTT NM_002111 .6 11412 AUGUUUGAGGAGGCCCUUAAGUCCGAUGUUUGAGGAGGCCCUUAAGGGAA HTT NM_002111 .6 11426CCUUAAGGGAAGCUACUGAA GAGGCCCUUAAGGGAAGCUACUGAAUUAUA HTT NM_002111 .611443 GAAUUAUAACACGUAAGAAA CUACUGAAUUAUAACACGUAAGAAAAUCAC HTTNM_002111 .6 11659 AUGUUUACAUUUGUAAGAAA GCUAGAUGUUUACAUUUGUAAGAAAUAACAHTT NM_002111 .6 11666 CAUUUGUAAGAAAUAACACUGUUUACAUUUGUAAGAAAUAACACUGUGAA HTT NM_002111 .6 11677AAUAACACUGUGAAUGUAAA UAAGAAAUAACACUGUGAAUGUAAAACAGA HTT NM_002111 .611863 AAUAUGAGCUCAUUAGUAAA AGAUGAAUAUGAGCUCAUUAGUAAAAAUGA HTTNM_002111 .6 11890 UCACCCACGCAUAUACAUAA UGACUUCAOCCACGCAUAUACAUAAAGUAUHTT NM_002111 .6 11927 AUAUAGACACAUCUAUAAUUUGUGCAUAUAGACACAUCUAUAAUUUUACA HTT NM_002111 .6 11947UUACACACACACCUCUCAAG UAAUUUUACACACACACCUCUCAAGACGGA HTT NM_002111 .612163 GACUUUAUCAUGUUCCUAAA AGGAAGACUUUAUCAUGUUCCUAAAAAUCU HTTNM_002111 .6 12218 UUGUUGCAAAUGUGAUUAAU AAAUUUUGUUGCAAAUGUGAUUAAUUUGGUHTT NM_002111 .6 12223 GCAAAUGUGAUUAAUUUGGUUUGUUGCAAAUGUGAUUAAUUUGGUUGUCA HTT NM_002111 .6 12235AAUUUGGUUGUCAAGUUUUG UGAUUAAUUUGGUUGUCAAGUUUUGGGGGU HTT NM_002111 .612279 UUUGUUUUCCUGCUGGUAAU UUGCUUUUGUUUUCCUGCUGGUAAUAUCGG HTTNM_002111 .6 12282 GUUUUCCUGCUGGUAAUAUC CUUUUGUUUUCCUGCUGGUAAUAUCGGGAAHTT NM_002111 .6 12297 AUAUCGGGAAAGAUUUUAAUUGGUAAUAUCGGGAAAGAUUUUAAUGAAAC HTT NM_002111 .6 12309AUUUUAAUGAAACCAGGGUA GAAAGAUUUUAAUGAAACCAGGGUAGAAUU HTT NM_002111 .612313 UAAUGAAACCAGGGUAGAAU GAUUUUAAUGAAACCAGGGUAGAAUUGUUU HTTNM_002111 .6 12331 AUUGUUUGGCAAUGCACUGA GUAGAAUUGUUUGGCAAUGCACUGAAGCGUHTT NM_002111 .6 13136 CCCCUCAGUUGUUUCUAAGAGCCUUCCCCUCAGUUGUUUCUAAGAGCAGA HTT NM_002111 .6 13398GGACUGACGAGAGAUGUAUA GGGAAGGACUGACGAGAGAUGUAUAUUUAA HTT NM_002111 .613403 GACGAGAGAUGUAUAUUUAA GGACUGACGAGAGAUGUAUAUUUAAUUUUU HTTNM_002111 .6 13423 UUUUUUAACUGCUGCAAACA UUUAAUUUUUUAACUGCUGCAAACAUUGUAHTT NM_002111 .6 13428 UAACUGCUGCAAACAUUGUAUUUUUUAACUGCUGCAAACAUUGUACAUCC HTT NM_002111 .6 152 ACCCUGGAAAAGCUGAUGAAUGGCGAOCCUGGAAAAGCUGAUGAAGGCCU HTT NM_002111 .6 170 AAGGCCUUCGAGUCCCUCAAUGAUGAAGGCCUUCGAGUCCCUCAAGUCCU HTT NM_002111 .6 402 CGCUGCACCGACCAAAGAAAGGAGCCGCUGCACCGACCAAAGAAAGAACU HTT NM_002111 .6 420 AAGAACUUUCAGCUACCAAGAAAGAAAGAACUUUCAGCUACCAAGAAAGA HTT NM_002111 .6 430 AGCUACCAAGAAAGACCGUGCUUUCAGCUACCAAGAAAGACCGUGUGAAU HTT NM_002111 .6 446 CGUGUGAAUCAUUGUCUGACAAGACCGUGUGAAUCAUUGUCUGACAAUAU HTT NM_002111 .6 454 UCAUUGUCUGACAAUAUGUGGUGAAUCAUUGUCUGACAAUAUGUGAAAAC HTT NM_002111 .6 462 UGACAAUAUGUGAAAACAUAUUGUCUGACAAUAUGUGAAAACAUAGUGGC HTT NM_002111 .6 467 AUAUGUGAAAACAUAGUGGCUGACAAUAUGUGAAAACAUAGUGGCACAGU HTT NM_002111 .6 211 GCAGCAGCAGCAGCAGCAGCCAGCAGCAGCAGCAGCAGCAGCAGCAGCAG

Table 1. Additional target sequences according to certain embodiments ofthe invention (SEQ ID NOS 5-212, respectively, in order of columns).

IV. siRNA Design

In some embodiments, siRNAs are designed as follows. First, a portion ofthe target gene (e.g., the htt gene), e.g., one or more of the targetsequences set forth at FIG. 8, is selected, e.g., 10150, 10146 and/or10125 from the 5′ untranslated region of a target gene. Cleavage of mRNAat these sites should eliminate translation of corresponding mutantprotein. Sense strands were designed based on the target sequence. (SeeFIG. 8.) Preferably the portion (and corresponding sense strand)includes about 19 to 25 nucleotides, e.g., 19, 20, 21, 22, 23, 24 or 25nucleotides. More preferably, the portion (and corresponding sensestrand) includes 21, 22 or 23 nucleotides. The skilled artisan willappreciate, however, that siRNAs having a length of less than 19nucleotides or greater than 25 nucleotides can also function to mediateRNAi. Accordingly, siRNAs of such length are also within the scope ofthe instant invention provided that they retain the ability to mediateRNAi. Longer RNAi agents have been demonstrated to elicit an interferonor PKR response in certain mammalian cells which may be undesirable.Preferably, the RNAi agents of the invention do not elicit a PKRresponse (i.e., are of a sufficiently short length). However, longerRNAi agents may be useful, for example, in cell types incapable ofgenerating a PRK response or in situations where the PKR response hasbeen down-regulated or dampened by alternative means.

The sense strand sequence is designed such that the target sequence isessentially in the middle of the strand. Moving the target sequence toan off-center position may, in some instances, reduce efficiency ofcleavage by the siRNA. Such compositions, i.e., less efficientcompositions, may be desirable for use if off-silencing of the wild-typemRNA is detected.

The antisense strand is routinely the same length as the sense strandand includes complementary nucleotides. In one embodiment, the strandsare fully complementary, i.e., the strands are blunt-ended when alignedor annealed. In another embodiment, the strands comprise align or annealsuch that 1-, 2-, 3-, 4-, 5-, 6- or 7-nucleotide overhangs aregenerated, i.e., the 3′ end of the sense strand extends 1, 2, 3, 4, 5, 6or 7 nucleotides further than the 5′ end of the antisense strand and/orthe 3′ end of the antisense strand extends 1, 2, 3, 4, 5, 6 or 7nucleotides further than the 5′ end of the sense strand. Overhangs cancomprise (or consist of) nucleotides corresponding to the target genesequence (or complement thereof). Alternatively, overhangs can comprise(or consist of) deoxyribonucleotides, for example dTs, or nucleotideanalogs, or other suitable non-nucleotide material.

To facilitate entry of the antisense strand into RISC (and thus increaseor improve the efficiency of target cleavage and silencing), the basepair strength between the 5′ end of the sense strand and 3′ end of theantisense strand can be altered, e.g., lessened or reduced, as describedin detail in U.S. Pat. Nos. 7,459,547, 7,772,203 and 7,732,593, entitled“Methods and Compositions for Controlling Efficacy of RNA Silencing”(filed Jun. 2, 2003) and U.S. Pat. Nos. 8,309,704, 7,750,144, 8,304,530,8,329,892 and 8,309,705, entitled “Methods and Compositions forEnhancing the Efficacy and Specificity of RNAi” (filed Jun. 2, 2003),the contents of which are incorporated in their entirety by thisreference. In one embodiment of these aspects of the invention, thebase-pair strength is less due to fewer G:C base pairs between the 5′end of the first or antisense strand and the 3′ end of the second orsense strand than between the 3′ end of the first or antisense strandand the 5′ end of the second or sense strand. In another embodiment, thebase pair strength is less due to at least one mismatched base pairbetween the 5′ end of the first or antisense strand and the 3′ end ofthe second or sense strand. In certain exemplary embodiments, themismatched base pair is selected from the group consisting of G:A, C:A,C:U, G:G, A:A, C:C and U:U. In another embodiment, the base pairstrength is less due to at least one wobble base pair, e.g., G:U,between the 5′ end of the first or antisense strand and the 3′ end ofthe second or sense strand. In another embodiment, the base pairstrength is less due to at least one base pair comprising a rarenucleotide, e.g., inosine (I). In certain exemplary embodiments, thebase pair is selected from the group consisting of an I:A, I:U and I:C.In yet another embodiment, the base pair strength is less due to atleast one base pair comprising a modified nucleotide. In certainexemplary embodiments, the modified nucleotide is selected from thegroup consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and2,6-diamino-A.

The design of siRNAs suitable for targeting the htt target sequences setforth at FIG. 8 is described in detail below. siRNAs can be designedaccording to the above exemplary teachings for any other targetsequences found in the htt gene. Moreover, the technology is applicableto targeting any other target sequences, e.g., non-disease causingtarget sequences.

To validate the effectiveness by which siRNAs destroy mRNAs (e.g.,huntingtin mRNA), the siRNA can be incubated with cDNA (e.g., huntingtincDNA) in a Drosophila-based in vitro mRNA expression system.Radiolabeled with ³²P, newly synthesized mRNAs (e.g., huntingtin mRNA)are detected autoradiographically on an agarose gel. The presence ofcleaved mRNA indicates mRNA nuclease activity. Suitable controls includeomission of siRNA. Alternatively, control siRNAs are selected having thesame nucleotide composition as the selected siRNA, but withoutsignificant sequence complementarity to the appropriate target gene.Such negative controls can be designed by randomly scrambling thenucleotide sequence of the selected siRNA; a homology search can beperformed to ensure that the negative control lacks homology to anyother gene in the appropriate genome. In addition, negative controlsiRNAs can be designed by introducing one or more base mismatches intothe sequence.

Sites of siRNA-mRNA complementation are selected which result in optimalmRNA specificity and maximal mRNA cleavage.

While the instant invention primarily features targeting specific targetsequences of a gene (e.g., in htt) distinct from the expanded CAG regionmutation, the skilled artisan will appreciate that targeting the mutantregion may have applicability as a therapeutic strategy in certainsituations. Targeting the mutant region can be accomplished using siRNAthat complements CAG in series. The siRNA^(cag) would bind to mRNAs withCAG complementation, but might be expected to have greater opportunityto bind to an extended CAG series. Multiple siRNA^(cag) would bind tothe mutant huntingtin mRNA (as opposed to fewer for the wild typehuntingtin mRNA); thus, the mutant huntingtin mRNA is more likely to becleaved. Successful mRNA inactivation using this approach would alsoeliminate normal or wild-type huntingtin mRNA. Also inactivated, atleast to some extent, could be other normal genes (approximately 70)which also have CAG repeats, where their mRNAs could interact with thesiRNA. This approach would thus rely on an attrition strategy—more ofthe mutant huntingtin mRNA would be destroyed than wild-type huntingtinmRNA or the other approximately 69 mRNAs that code for polyglutamines.

V. RNAi Agents

The present invention includes siRNA molecules designed, for example, asdescribed above. The siRNA molecules of the invention can be chemicallysynthesized, or can be transcribed in vitro from a DNA template, or invivo from e.g., shRNA, or by using recombinant human DICER enzyme, tocleave in vitro transcribed dsRNA templates into pools of 20-, 21- or23-bp duplex RNA mediating RNAi. The siRNA molecules can be designedusing any method known in the art.

In one aspect, instead of the RNAi agent being an interferingribonucleic acid, e.g., an siRNA or shRNA as described above, the RNAiagent can encode an interfering ribonucleic acid, e.g., an shRNA, asdescribed above. In other words, the RNAi agent can be a transcriptionaltemplate of the interfering ribonucleic acid. Thus, RNAi agents of thepresent invention can also include small hairpin RNAs (shRNAs), andexpression constructs engineered to express shRNAs. Transcription ofshRNAs is initiated at a polymerase III (pol III) promoter, and isthought to be terminated at position 2 of a 4-5-thymine transcriptiontermination site. Upon expression, shRNAs are thought to fold into astem-loop structure with 3′ UU-overhangs; subsequently, the ends ofthese shRNAs are processed, converting the shRNAs into siRNA-likemolecules of about 21-23 nucleotides (Brummelkamp et al., 2002; Lee etal., 2002, Supra; Miyagishi et al., 2002; Paddison et al., 2002, supra;Paul et al., 2002, supra; Sui et al., 2002 supra; Yu et al., 2002,supra. More information about shRNA design and use can be found on theinterne at the following addresses:katandin.cshl.org:9331/RNAi/docs/BseRI-BamHI_Strategy.pdf andkatandin.cshl.org:9331/RNAi/docs/Web_version_of_PCR_strategy1.pdf).

Expression constructs of the present invention include any constructsuitable for use in the appropriate expression system and include, butare not limited to, retroviral vectors, linear expression cassettes,plasmids and viral or virally-derived vectors, as known in the art. Suchexpression constructs can include one or more inducible promoters, RNAPol III promoter systems such as U6 snRNA promoters or H1 RNA polymeraseIII promoters, or other promoters known in the art. The constructs caninclude one or both strands of the siRNA. Expression constructsexpressing both strands can also include loop structures linking bothstrands, or each strand can be separately transcribed from separatepromoters within the same construct. Each strand can also be transcribedfrom a separate expression construct. (Tuschl, T., 2002, Supra).

Synthetic siRNAs can be delivered into cells by methods known in theart, including cationic liposome transfection and electroporation. Toobtain longer term suppression of the target genes (i.e., htt genes) andto facilitate delivery under certain circumstances, one or more siRNAcan be expressed within cells from recombinant DNA constructs. Suchmethods for expressing siRNA duplexes within cells from recombinant DNAconstructs to allow longer-term target gene suppression in cells areknown in the art, including mammalian Pol III promoter systems (e.g., H1or U6/snRNA promoter systems (Tuschl, T., 2002, supra) capable ofexpressing functional double-stranded siRNAs; (Bagella et al., 1998; Leeet al., 2002, supra; Miyagishi et al., 2002, supra; Paul et al., 2002,supra; Yu et al., 2002), supra; Sui et al., 2002, supra).Transcriptional termination by RNA Pol III occurs at runs of fourconsecutive T residues in the DNA template, providing a mechanism to endthe siRNA transcript at a specific sequence. The siRNA is complementaryto the sequence of the target gene in 5′-3′ and 3′-5′ orientations, andthe two strands of the siRNA can be expressed in the same construct orin separate constructs. Hairpin siRNAs, driven by H1 or U6 snRNApromoter and expressed in cells, can inhibit target gene expression(Bagella et al., 1998; Lee et al., 2002, supra; Miyagishi et al., 2002,supra; Paul et al., 2002, supra; Yu et al., 2002), supra; Sui et al.,2002, supra). Constructs containing siRNA sequence under the control ofT7 promoter also make functional siRNAs when cotransfected into thecells with a vector expressing T7 RNA polymerase (Jacque et al., 2002,supra). A single construct may contain multiple sequences coding forsiRNAs, such as multiple regions of the gene encoding htt, targeting thesame gene or multiple genes, and can be driven, for example, by separatePolIII promoter sites.

Animal cells express a range of noncoding RNAs of approximately 22nucleotides termed micro RNA (miRNAs) which can regulate gene expressionat the post transcriptional or translational level during animaldevelopment. One common feature of miRNAs is that they are all excisedfrom an approximately 70 nucleotide precursor RNA stem-loop, probably byDicer, an RNase III-type enzyme, or a homolog thereof. By substitutingthe stem sequences of the miRNA precursor with sequence complementary tothe target mRNA, a vector construct that expresses the engineeredprecursor can be used to produce siRNAs to initiate RNAi againstspecific mRNA targets in mammalian cells (Zeng et al., 2002, supra).When expressed by DNA vectors containing polymerase III promoters,micro-RNA designed hairpins can silence gene expression (McManus et al.,2002, supra). MicroRNAs targeting polymorphisms may also be useful forblocking translation of mutant proteins, in the absence ofsiRNA-mediated gene-silencing. Such applications may be useful insituations, for example, where a designed siRNA caused off-targetsilencing of wild type protein.

Viral-mediated delivery mechanisms can also be used to induce specificsilencing of targeted genes through expression of siRNA, for example, bygenerating recombinant adenoviruses harboring siRNA under RNA Pol IIpromoter transcription control (Xia et al., 2002, supra). Infection ofHeLa cells by these recombinant adenoviruses allows for diminishedendogenous target gene expression. Injection of the recombinantadenovirus vectors into transgenic mice expressing the target genes ofthe siRNA results in in vivo reduction of target gene expression. Id. Inan animal model, whole-embryo electroporation can efficiently deliversynthetic siRNA into post-implantation mouse embryos (Calegari et al.,2002). In adult mice, efficient delivery of siRNA can be accomplished by“high-pressure” delivery technique, a rapid injection (within 5 seconds)of a large volume of siRNA containing solution into animal via the tailvein (Liu et al., 1999, supra; McCaffrey et al., 2002, supra; Lewis etal., 2002. Nanoparticles and liposomes can also be used to deliver siRNAinto animals. In certain exemplary embodiments, recombinantadeno-associated viruses (rAAVs) and their associated vectors can beused to deliver one or more siRNAs into cells, e.g., neural cells (e.g.,brain cells) (US Patent Applications 2014/0296486, 2010/0186103,2008/0269149, 2006/0078542 and 2005/0220766).

The nucleic acid compositions of the invention include both unmodifiedsiRNAs and modified siRNAs as known in the art, such as crosslinkedsiRNA derivatives or derivatives having non nucleotide moieties linked,for example to their 3′ or 5′ ends. Modifying siRNA derivatives in thisway may improve cellular uptake or enhance cellular targeting activitiesof the resulting siRNA derivative as compared to the correspondingsiRNA, are useful for tracing the siRNA derivative in the cell, orimprove the stability of the siRNA derivative compared to thecorresponding siRNA.

Engineered RNA precursors, introduced into cells or whole organisms asdescribed herein, will lead to the production of a desired siRNAmolecule. Such an siRNA molecule will then associate with endogenousprotein components of the RNAi pathway to bind to and target a specificmRNA sequence for cleavage and destruction. In this fashion, the mRNA tobe targeted by the siRNA generated from the engineered RNA precursorwill be depleted from the cell or organism, leading to a decrease in theconcentration of the protein encoded by that mRNA in the cell ororganism. The RNA precursors are typically nucleic acid molecules thatindividually encode either one strand of a dsRNA or encode the entirenucleotide sequence of an RNA hairpin loop structure.

The nucleic acid compositions of the invention can be unconjugated orcan be conjugated to another moiety, such as a nanoparticle, to enhancea property of the compositions, e.g., a pharmacokinetic parameter suchas absorption, efficacy, bioavailability and/or half-life. Theconjugation can be accomplished by methods known in the art, e.g., usingthe methods of Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001)(describes nucleic acids loaded to polyalkylcyanoacrylate (PACA)nanoparticles); Fattal et al., J. Control Release 53(1-3):137-43 (1998)(describes nucleic acids bound to nanoparticles); Schwab et al., Ann.Oncol. 5 Suppl. 4:55-8 (1994) (describes nucleic acids linked tointercalating agents, hydrophobic groups, polycations or PACAnanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10 (1995)(describes nucleic acids linked to nanoparticles).

The nucleic acid molecules of the present invention can also be labeledusing any method known in the art. For instance, the nucleic acidcompositions can be labeled with a fluorophore, e.g., Cy3, fluorescein,or rhodamine. The labeling can be carried out using a kit, e.g., theSILENCER™ siRNA labeling kit (Ambion). Additionally, the siRNA can beradiolabeled, e.g., using ³H, ³²P or other appropriate isotope.

Moreover, because RNAi is believed to progress via at least onesingle-stranded RNA intermediate, the skilled artisan will appreciatethat ss-siRNAs (e.g., the antisense strand of a ds-siRNA) can also bedesigned (e.g., for chemical synthesis) generated (e.g., enzymaticallygenerated) or expressed (e.g., from a vector or plasmid) as describedherein and utilized according to the claimed methodologies. Moreover, ininvertebrates, RNAi can be triggered effectively by long dsRNAs (e.g.,dsRNAs about 100-1000 nucleotides in length, preferably about 200-500,for example, about 250, 300, 350, 400 or 450 nucleotides in length)acting as effectors of RNAi. (Brondani et al., Proc Natl Acad Sci USA.2001 Dec. 4; 98(25):14428-33. Epub 2001 Nov. 27.)

VI. Anti-Htt RNA Silencing Agents

The present invention features anti-huntingtin RNA silencing agents(e.g., siRNA and shRNAs), methods of making said RNA silencing agents,and methods (e.g., research and/or therapeutic methods) for using saidimproved RNA silencing agents (or portions thereof) for RNA silencing ofhuntingtin protein (e.g., mutant huntingtin protein). The RNA silencingagents comprise an antisense strand (or portions thereof), wherein theantisense strand has sufficient complementary to a heterozygous singlenucleotide polymorphism to mediate an RNA-mediated silencing mechanism(e.g. RNAi).

In certain embodiments, siRNA compounds are provided having one or anycombination of the following properties: (1) fully chemically-stabilized(i.e., no unmodified 2′-OH residues); (2) asymmetry; (3) 11-16 base pairduplexes; (4) alternating pattern of chemically-modified nucleotides(e.g., 2′-fluoro and 2′-methoxy modifications); and (5) single-stranded,fully phosphorothioated tails of 5-8 bases. The number ofphosphorothioate modifications is critical. This number is varied from 6to 17 total in different embodiments.

In certain embodiments, the siRNA compounds described herein can beconjugated to a variety of targeting agents, including, but not limitedto, cholesterol, DHA, phenyltropanes, cortisol, vitamin A, vitamin D,GalNac, and gangliozides. The cholesterol-modified version showed 5-10fold improvement in efficacy in vitro versus previously used chemicalstabilization patterns (e.g., wherein all purine but not purimidines aremodified) in wide range of cell types (e.g., HeLa, neurons, hepatocytes,trophoblasts).

Certain compounds of the invention having the structural propertiesdescribed above and herein may be referred to as “hsiRNA-ASP”(hydrophobically-modified, small interfering RNA, featuring an advancedstabilization pattern). In addition, this hsiRNA-ASP pattern showed adramatically improved distribution through the brain, spinal cord,delivery to liver, placenta, kidney, spleen and several other tissues,making them accessible for therapeutic intervention.

In liver hsiRNA-ASP delivery specifically to endothelial and kuppercells, but not hepatocytes, making this chemical modification patterncomplimentary rather than competitive technology to GalNac conjugates.

The compounds of the invention can be described in the following aspectsand embodiments.

In a first aspect, provided herein is oligonucleotide of at least 16contiguous nucleotides, said oligonucleotide having a 5′ end, a 3′ endand complementarity to a target, wherein: (1) the oligonucleotidecomprises alternating 2′-methoxy-ribonucleotides and2′-fluoro-ribonucleotides; (2) the nucleotides at positions 2 and 14from the 5′ end are not 2′-methoxy-ribonucleotides; (3) the nucleotidesare connected via phosphodiester or phosphorothioate linkages; and (4)the nucleotides at positions 1-6 from the 3′ end, or positions 1-7 fromthe 3′ end, are connected to adjacent nucleotides via phosphorothioatelinkages.

In a second aspect, provided herein is a double-stranded,chemically-modified nucleic acid, comprising a first oligonucleotide anda second oligonucleotide, wherein: (1) the first oligonucleotide is anoligonucleotide described herein (e.g., comprising SEQ ID Nos:1, 2, 3 or4); (2) a portion of the first oligonucleotide is complementary to aportion of the second oligonucleotide; (3) the second oligonucleotidecomprises alternating 2′-methoxy-ribonucleotides and2′-fluoro-ribonucleotides; (4) the nucleotides at positions 2 and 14from the 3′ end of the second oligonucleotide are2′-methoxy-ribonucleotides; and (5) the nucleotides of the secondoligonucleotide are connected via phosphodiester or phosphorothioatelinkages.

In a third aspect, provided herein is oligonucleotide having thestructure:

X-A(-L-B-L-A)j (-S-B-S-A)r(-S-B)t-OR

wherein: X is a 5′ phosphate group; A, for each occurrence,independently is a 2′-methoxy-ribonucleotide; B, for each occurrence,independently is a 2′-fluoro-ribonucleotide; L, for each occurrenceindependently is a phosphodiester or phosphorothioate linker; S is aphosphorothioate linker; and R is selected from hydrogen and a cappinggroup (e.g., an acyl such as acetyl); j is 4, 5, 6 or 7; r is 2 or 3;and t is 0 or 1.

In a fourth aspect, provided herein is a double-stranded,chemically-modified nucleic acid comprising a first oligonucleotide anda second oligonucleotide, wherein: (1) the first oligonucleotide isselected from the oligonucleotides of the third aspect; (2) a portion ofthe first oligonucleotide is complementary to a portion of the secondoligonucleotide; and (3) the second oligonucleotide has the structure:

C-L-B(-S-A-S-B)m′(-P-A-P-B)n′(-P-A-S-B)q′(-S-A)r′(-S-B)t′-OR

wherein: C is a hydrophobic molecule; A, for each occurrence,independently is a 2′-methoxy-ribonucleotide; B, for each occurrence,independently is a 2′-fluoro-ribonucleotide; L is a linker comprisingone or more moiety selected from the group consisting of: 0-4 repeatunits of ethyleneglycol, a phosphodiester, and a phosphorothioate; S isa phosphorothioate linker; P is a phosphodiester linker; R is selectedfrom hydrogen and a capping group (e.g., an acyl such as acetyl); m′ is0 or 1; n′ is 4, 5 or 6; q′ is 0 or 1; r′ is 0 or 1; and t′ is 0 or 1.

a) Design of Anti-Htt siRNA Molecules

An siRNA molecule of the invention is a duplex consisting of a sensestrand and complementary antisense strand, the antisense strand havingsufficient complementary to an htt mRNA to mediate RNAi. Preferably, thesiRNA molecule has a length from about 10-50 or more nucleotides, i.e.,each strand comprises 10-50 nucleotides (or nucleotide analogs). Morepreferably, the siRNA molecule has a length from about 16-30, e.g., 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides ineach strand, wherein one of the strands is sufficiently complementary toa target region. Preferably, the strands are aligned such that there areat least 1, 2, or 3 bases at the end of the strands which do not align(i.e., for which no complementary bases occur in the opposing strand)such that an overhang of 1, 2 or 3 residues occurs at one or both endsof the duplex when strands are annealed. Preferably, the siRNA moleculehas a length from about 10-50 or more nucleotides, i.e., each strandcomprises 10-50 nucleotides (or nucleotide analogs). More preferably,the siRNA molecule has a length from about 16-30, e.g., 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in eachstrand, wherein one of the strands is substantially complementary to atarget sequence, and the other strand is identical or substantiallyidentical to the first strand.

Generally, siRNAs can be designed by using any method known in the art,for instance, by using the following protocol:

1. The siRNA should be specific for a target sequence, e.g., a targetsequence set forth in FIG. 8. In one embodiment, a target sequence isfound in a mutant huntingtin (htt) allele, but not a wild-typehuntingtin allele. In another embodiment, a target sequence is found inboth a mutant huntingtin (htt) allele, and a wild-type huntingtinallele. In another embodiment, a target sequence is found in a wild-typehuntingtin allele. The first strand should be complementary to thetarget sequence, and the other strand is substantially complementary tothe first strand. (See FIG. 8 for exemplary sense and antisensestrands.) In one embodiment, the target sequence is outside the expandedCAG repeat of the mutant huntingin (htt) allele. In another embodiment,the target sequence is outside a coding region of the target gene.Exemplary target sequences are selected from the 5′ untranslated region(5′-UTR) of a target gene. Cleavage of mRNA at these sites shouldeliminate translation of corresponding mutant protein. Target sequencesfrom other regions of the htt gene are also suitable for targeting. Asense strand is designed based on the target sequence. Further, siRNAswith lower G/C content (35-55%) may be more active than those with G/Ccontent higher than 55%. Thus in one embodiment, the invention includesnucleic acid molecules having 35-55% G/C content.

2. The sense strand of the siRNA is designed based on the sequence ofthe selected target site. Preferably the sense strand includes about 19to 25 nucleotides, e.g., 19, 20, 21, 22, 23, 24 or 25 nucleotides. Morepreferably, the sense strand includes 21, 22 or 23 nucleotides. Theskilled artisan will appreciate, however, that siRNAs having a length ofless than 19 nucleotides or greater than 25 nucleotides can alsofunction to mediate RNAi. Accordingly, siRNAs of such length are alsowithin the scope of the instant invention provided that they retain theability to mediate RNAi. Longer RNA silencing agents have beendemonstrated to elicit an interferon or Protein Kinase R (PKR) responsein certain mammalian cells which may be undesirable. Preferably the RNAsilencing agents of the invention do not elicit a PKR response (i.e.,are of a sufficiently short length). However, longer RNA silencingagents may be useful, for example, in cell types incapable of generatinga PRK response or in situations where the PKR response has beendown-regulated or dampened by alternative means.

The siRNA molecules of the invention have sufficient complementaritywith the target sequence such that the siRNA can mediate RNAi. Ingeneral, siRNA containing nucleotide sequences sufficiently identical toa target sequence portion of the target gene to effect RISC-mediatedcleavage of the target gene are preferred. Accordingly, in a preferredembodiment, the sense strand of the siRNA is designed have to have asequence sufficiently identical to a portion of the target. For example,the sense strand may have 100% identity to the target site. However,100% identity is not required. Greater than 80% identity, e.g., 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or even 100% identity, between the sense strandand the target RNA sequence is preferred. The invention has theadvantage of being able to tolerate certain sequence variations toenhance efficiency and specificity of RNAi. In one embodiment, the sensestrand has 4, 3, 2, 1, or 0 mismatched nucleotide(s) with a targetregion, such as a target region that differs by at least one base pairbetween a wild-type and mutant allele, e.g., a target region comprisingthe gain-of-function mutation, and the other strand is identical orsubstantially identical to the first strand. Moreover, siRNA sequenceswith small insertions or deletions of 1 or 2 nucleotides may also beeffective for mediating RNAi. Alternatively, siRNA sequences withnucleotide analog substitutions or insertions can be effective forinhibition.

Sequence identity may be determined by sequence comparison and alignmentalgorithms known in the art. To determine the percent identity of twonucleic acid sequences (or of two amino acid sequences), the sequencesare aligned for optimal comparison purposes (e.g., gaps can beintroduced in the first sequence or second sequence for optimalalignment). The nucleotides (or amino acid residues) at correspondingnucleotide (or amino acid) positions are then compared. When a positionin the first sequence is occupied by the same residue as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=number of identical positions/totalnumber of positions×100), optionally penalizing the score for the numberof gaps introduced and/or length of gaps introduced.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In one embodiment, the alignment generated over a certainportion of the sequence aligned having sufficient identity but not overportions having low degree of identity (i.e., a local alignment). Apreferred, non-limiting example of a local alignment algorithm utilizedfor the comparison of sequences is the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithmis incorporated into the BLAST programs (version 2.0) of Altschul, etal. (1990) J. Mol. Biol. 215:403-10.

In another embodiment, the alignment is optimized by introducingappropriate gaps and percent identity is determined over the length ofthe aligned sequences (i.e., a gapped alignment). To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. In another embodiment, the alignment is optimized byintroducing appropriate gaps and percent identity is determined over theentire length of the sequences aligned (i.e., a global alignment). Apreferred, non-limiting example of a mathematical algorithm utilized forthe global comparison of sequences is the algorithm of Myers and Miller,CABIOS (1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

3. The antisense or guide strand of the siRNA is routinely the samelength as the sense strand and includes complementary nucleotides. Inone embodiment, the guide and sense strands are fully complementary,i.e., the strands are blunt-ended when aligned or annealed. In anotherembodiment, the strands of the siRNA can be paired in such a way as tohave a 3′ overhang of 1 to 7 (e.g., 2, 3, 4, 5, 6 or 7), or 1 to 4,e.g., 2, 3 or 4 nucleotides. Overhangs can comprise (or consist of)nucleotides corresponding to the target gene sequence (or complementthereof). Alternatively, overhangs can comprise (or consist of)deoxyribonucleotides, for example dTs, or nucleotide analogs, or othersuitable non-nucleotide material. Thus in another embodiment, thenucleic acid molecules may have a 3′ overhang of 2 nucleotides, such asTT. The overhanging nucleotides may be either RNA or DNA. As notedabove, it is desirable to choose a target region wherein the mutant:wildtype mismatch is a purine:purine mismatch.

4. Using any method known in the art, compare the potential targets tothe appropriate genome database (human, mouse, rat, etc.) and eliminatefrom consideration any target sequences with significant homology toother coding sequences. One such method for such sequence homologysearches is known as BLAST, which is available at National Center forBiotechnology Information website.

5. Select one or more sequences that meet your criteria for evaluation.

Further general information about the design and use of siRNA may befound in “The siRNA User Guide,” available at The Max-Plank-Institut furBiophysikalishe Chemie website.

Alternatively, the siRNA may be defined functionally as a nucleotidesequence (or oligonucleotide sequence) that is capable of hybridizingwith the target sequence (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. hybridization for 12-16 hours; followed bywashing). Additional preferred hybridization conditions includehybridization at 70° C. in 1×SSC or 50° C. in 1×SSC, 50% formamidefollowed by washing at 70° C. in 0.3×SSC or hybridization at 70° C. in4×SSC or 50° C. in 4×SSC, 50% formamide followed by washing at 67° C. in1×SSC. The hybridization temperature for hybrids anticipated to be lessthan 50 base pairs in length should be 5-10° C. less than the meltingtemperature (Tm) of the hybrid, where Tm is determined according to thefollowing equations. For hybrids less than 18 base pairs in length, Tm(°C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49base pairs in length, Tm(° C.)=81.5+16.6(log 10[Na+])+0.41(%G+C)−(600/N), where N is the number of bases in the hybrid, and [Na+] isthe concentration of sodium ions in the hybridization buffer ([Na+] for1×SSC=0.165 M). Additional examples of stringency conditions forpolynucleotide hybridization are provided in Sambrook, J., E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters9 and 11, and Current Protocols in Molecular Biology, 1995, F. M.Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and6.3-6.4, incorporated herein by reference.

Negative control siRNAs should have the same nucleotide composition asthe selected siRNA, but without significant sequence complementarity tothe appropriate genome. Such negative controls may be designed byrandomly scrambling the nucleotide sequence of the selected siRNA. Ahomology search can be performed to ensure that the negative controllacks homology to any other gene in the appropriate genome. In addition,negative control siRNAs can be designed by introducing one or more basemismatches into the sequence.

6. To validate the effectiveness by which siRNAs destroy target mRNAs(e.g., wild-type or mutant huntingtin mRNA), the siRNA may be incubatedwith target cDNA (e.g., huntingtin cDNA) in a Drosophila-based in vitromRNA expression system. Radiolabeled with ³²P, newly synthesized targetmRNAs (e.g., huntingtin mRNA) are detected autoradiographically on anagarose gel. The presence of cleaved target mRNA indicates mRNA nucleaseactivity. Suitable controls include omission of siRNA and use ofnon-target cDNA. Alternatively, control siRNAs are selected having thesame nucleotide composition as the selected siRNA, but withoutsignificant sequence complementarity to the appropriate target gene.Such negative controls can be designed by randomly scrambling thenucleotide sequence of the selected siRNA. A homology search can beperformed to ensure that the negative control lacks homology to anyother gene in the appropriate genome. In addition, negative controlsiRNAs can be designed by introducing one or more base mismatches intothe sequence.

Anti-htt siRNAs may be designed to target any of the target sequencesdescribed supra. Said siRNAs comprise an antisense strand which issufficiently complementary with the target sequence to mediate silencingof the target sequence. In certain embodiments, the RNA silencing agentis a siRNA.

In certain embodiments, the siRNA comprises a sense strand comprising asequence set forth at FIG. 8, and an antisense strand comprising asequence set forth at FIG. 8.

Sites of siRNA-mRNA complementation are selected which result in optimalmRNA specificity and maximal mRNA cleavage.

b) siRNA-Like Molecules

siRNA-like molecules of the invention have a sequence (i.e., have astrand having a sequence) that is “sufficiently complementary” to atarget sequence of a htt mRNA to direct gene silencing either by RNAi ortranslational repression. siRNA-like molecules are designed in the sameway as siRNA molecules, but the degree of sequence identity between thesense strand and target RNA approximates that observed between an miRNAand its target. In general, as the degree of sequence identity between amiRNA sequence and the corresponding target gene sequence is decreased,the tendency to mediate post-transcriptional gene silencing bytranslational repression rather than RNAi is increased. Therefore, in analternative embodiment, where post-transcriptional gene silencing bytranslational repression of the target gene is desired, the miRNAsequence has partial complementarity with the target gene sequence. Incertain embodiments, the miRNA sequence has partial complementarity withone or more short sequences (complementarity sites) dispersed within thetarget mRNA (e.g. within the 3′-UTR of the target mRNA) (Hutvagner andZamore, Science, 2002; Zeng et al., Mol. Cell, 2002; Zeng et al., RNA,2003; Doench et al., Genes & Dev., 2003). Since the mechanism oftranslational repression is cooperative, multiple complementarity sites(e.g., 2, 3, 4, 5, or 6) may be targeted in certain embodiments.

The capacity of a siRNA-like duplex to mediate RNAi or translationalrepression may be predicted by the distribution of non-identicalnucleotides between the target gene sequence and the nucleotide sequenceof the silencing agent at the site of complementarity. In oneembodiment, where gene silencing by translational repression is desired,at least one non-identical nucleotide is present in the central portionof the complementarity site so that duplex formed by the miRNA guidestrand and the target mRNA contains a central “bulge” (Doench J G etal., Genes & Dev., 2003). In another embodiment 2, 3, 4, 5, or 6contiguous or non-contiguous non-identical nucleotides are introduced.The non-identical nucleotide may be selected such that it forms a wobblebase pair (e.g., G:U) or a mismatched base pair (G:A, C:A, C:U, G:G,A:A, C:C, U:U). In a further preferred embodiment, the “bulge” iscentered at nucleotide positions 12 and 13 from the 5′ end of the miRNAmolecule.

c) Short Hairpin RNA (shRNA) Molecules

In certain featured embodiments, the instant invention provides shRNAscapable of mediating RNA silencing of an htt target sequence withenhanced selectivity. In contrast to siRNAs, shRNAs mimic the naturalprecursors of micro RNAs (miRNAs) and enter at the top of the genesilencing pathway. For this reason, shRNAs are believed to mediate genesilencing more efficiently by being fed through the entire natural genesilencing pathway.

miRNAs are noncoding RNAs of approximately 22 nucleotides which canregulate gene expression at the post transcriptional or translationallevel during plant and animal development. One common feature of miRNAsis that they are all excised from an approximately 70 nucleotideprecursor RNA stem-loop termed pre-miRNA, probably by Dicer, an RNaseIII-type enzyme, or a homolog thereof. Naturally-occurring miRNAprecursors (pre-miRNA) have a single strand that forms a duplex stemincluding two portions that are generally complementary, and a loop,that connects the two portions of the stem. In typical pre-miRNAs, thestem includes one or more bulges, e.g., extra nucleotides that create asingle nucleotide “loop” in one portion of the stem, and/or one or moreunpaired nucleotides that create a gap in the hybridization of the twoportions of the stem to each other. Short hairpin RNAs, or engineeredRNA precursors, of the invention are artificial constructs based onthese naturally occurring pre-miRNAs, but which are engineered todeliver desired RNA silencing agents (e.g., siRNAs of the invention). Bysubstituting the stem sequences of the pre-miRNA with sequencecomplementary to the target mRNA, a shRNA is formed. The shRNA isprocessed by the entire gene silencing pathway of the cell, therebyefficiently mediating RNAi.

The requisite elements of a shRNA molecule include a first portion and asecond portion, having sufficient complementarity to anneal or hybridizeto form a duplex or double-stranded stem portion. The two portions neednot be fully or perfectly complementary. The first and second “stem”portions are connected by a portion having a sequence that hasinsufficient sequence complementarity to anneal or hybridize to otherportions of the shRNA. This latter portion is referred to as a “loop”portion in the shRNA molecule. The shRNA molecules are processed togenerate siRNAs. shRNAs can also include one or more bulges, i.e., extranucleotides that create a small nucleotide “loop” in a portion of thestem, for example a one-, two- or three-nucleotide loop. The stemportions can be the same length, or one portion can include an overhangof, for example, 1-5 nucleotides. The overhanging nucleotides caninclude, for example, uracils (Us), e.g., all Us. Such Us are notablyencoded by thymidines (Ts) in the shRNA-encoding DNA which signal thetermination of transcription.

In shRNAs (or engineered precursor RNAs) of the instant invention, oneportion of the duplex stem is a nucleic acid sequence that iscomplementary (or anti-sense) to the htt target sequence. Preferably,one strand of the stem portion of the shRNA is sufficientlycomplementary (e.g., antisense) to a target RNA (e.g., mRNA) sequence tomediate degradation or cleavage of said target RNA via RNA interference(RNAi). Thus, engineered RNA precursors include a duplex stem with twoportions and a loop connecting the two stem portions. The antisenseportion can be on the 5′ or 3′ end of the stem. The stem portions of ashRNA are preferably about 15 to about 50 nucleotides in length.Preferably the two stem portions are about 18 or 19 to about 21, 22, 23,24, 25, 30, 35, 37, 38, 39, or 40 or more nucleotides in length. Inpreferred embodiments, the length of the stem portions should be 21nucleotides or greater. When used in mammalian cells, the length of thestem portions should be less than about 30 nucleotides to avoidprovoking non-specific responses like the interferon pathway. Innon-mammalian cells, the stem can be longer than 30 nucleotides. Infact, the stem can include much larger sections complementary to thetarget mRNA (up to, and including the entire mRNA). In fact, a stemportion can include much larger sections complementary to the targetmRNA (up to, and including the entire mRNA).

The two portions of the duplex stem must be sufficiently complementaryto hybridize to form the duplex stem. Thus, the two portions can be, butneed not be, fully or perfectly complementary. In addition, the two stemportions can be the same length, or one portion can include an overhangof 1, 2, 3, or 4 nucleotides. The overhanging nucleotides can include,for example, uracils (Us), e.g., all Us. The loop in the shRNAs orengineered RNA precursors may differ from natural pre-miRNA sequences bymodifying the loop sequence to increase or decrease the number of pairednucleotides, or replacing all or part of the loop sequence with atetraloop or other loop sequences. Thus, the loop in the shRNAs orengineered RNA precursors can be 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g.,15 or 20, or more nucleotides in length.

The loop in the shRNAs or engineered RNA precursors may differ fromnatural pre-miRNA sequences by modifying the loop sequence to increaseor decrease the number of paired nucleotides, or replacing all or partof the loop sequence with a tetraloop or other loop sequences. Thus, theloop portion in the shRNA can be about 2 to about 20 nucleotides inlength, i.e., about 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g., 15 or 20, ormore nucleotides in length. A preferred loop consists of or comprises a“tetraloop” sequences. Exemplary tetraloop sequences include, but arenot limited to, the sequences GNRA, where N is any nucleotide and R is apurine nucleotide, GGGG, and UUUU.

In certain embodiments, shRNAs of the invention include the sequences ofa desired siRNA molecule described supra. In other embodiments, thesequence of the antisense portion of a shRNA can be designed essentiallyas described above or generally by selecting an 18, 19, 20, 21nucleotide, or longer, sequence from within the target RNA (e.g., httmRNA), for example, from a region 100 to 200 or 300 nucleotides upstreamor downstream of the start of translation. In general, the sequence canbe selected from any portion of the target RNA (e.g., mRNA) includingthe 5′ UTR (untranslated region), coding sequence, or 3′ UTR, providedsaid portion is distant from the site of the gain-of-function mutation.This sequence can optionally follow immediately after a region of thetarget gene containing two adjacent AA nucleotides. The last twonucleotides of the nucleotide sequence can be selected to be UU. This 21or so nucleotide sequence is used to create one portion of a duplex stemin the shRNA. This sequence can replace a stem portion of a wild-typepre-miRNA sequence, e.g., enzymatically, or is included in a completesequence that is synthesized. For example, one can synthesize DNAoligonucleotides that encode the entire stem-loop engineered RNAprecursor, or that encode just the portion to be inserted into theduplex stem of the precursor, and using restriction enzymes to build theengineered RNA precursor construct, e.g., from a wild-type pre-miRNA.

Engineered RNA precursors include in the duplex stem the 21-22 or sonucleotide sequences of the siRNA or siRNA-like duplex desired to beproduced in vivo. Thus, the stem portion of the engineered RNA precursorincludes at least 18 or 19 nucleotide pairs corresponding to thesequence of an exonic portion of the gene whose expression is to bereduced or inhibited. The two 3′ nucleotides flanking this region of thestem are chosen so as to maximize the production of the siRNA from theengineered RNA precursor and to maximize the efficacy of the resultingsiRNA in targeting the corresponding mRNA for translational repressionor destruction by RNAi in vivo and in vitro.

In certain embodiments, shRNAs of the invention include miRNA sequences,optionally end-modified miRNA sequences, to enhance entry into RISC. ThemiRNA sequence can be similar or identical to that of any naturallyoccurring miRNA (see e.g. The miRNA Registry; Griffiths-Jones S, Nuc.Acids Res., 2004). Over one thousand natural miRNAs have been identifiedto date and together they are thought to comprise about 1% of allpredicted genes in the genome. Many natural miRNAs are clusteredtogether in the introns of pre-mRNAs and can be identified in silicousing homology-based searches (Pasquinelli et al., 2000; Lagos-Quintanaet al., 2001; Lau et al., 2001; Lee and Ambros, 2001) or computeralgorithms (e.g. MiRScan, MiRSeeker) that predict the capability of acandidate miRNA gene to form the stem loop structure of a pri-mRNA (Gradet al., Mol. Cell., 2003; Lim et al., Genes Dev., 2003; Lim et al.,Science, 2003; Lai E C et al., Genome Bio., 2003). An online registryprovides a searchable database of all published miRNA sequences (ThemiRNA Registry at the Sanger Institute website; Griffiths-Jones S, Nuc.Acids Res., 2004). Exemplary, natural miRNAs include lin-4, let-7,miR-10, mirR-15, miR-16, miR-168, miR-175, miR-196 and their homologs,as well as other natural miRNAs from humans and certain model organismsincluding Drosophila melanogaster, Caenorhabditis elegans, zebrafish,Arabidopsis thalania, Mus musculus, and Rattus norvegicus as describedin International PCT Publication No. WO 03/029459.

Naturally-occurring miRNAs are expressed by endogenous genes in vivo andare processed from a hairpin or stem-loop precursor (pre-miRNA orpri-miRNAs) by Dicer or other RNAses (Lagos-Quintana et al., Science,2001; Lau et al., Science, 2001; Lee and Ambros, Science, 2001;Lagos-Quintana et al., Curr. Biol., 2002; Mourelatos et al., Genes Dev.,2002; Reinhart et al., Science, 2002; Ambros et al., Curr. Biol., 2003;Brennecke et al., 2003; Lagos-Quintana et al., RNA, 2003; Lim et al.,Genes Dev., 2003; Lim et al., Science, 2003). miRNAs can existtransiently in vivo as a double-stranded duplex, but only one strand istaken up by the RISC complex to direct gene silencing. Certain miRNAs,e.g., plant miRNAs, have perfect or near-perfect complementarity totheir target mRNAs and, hence, direct cleavage of the target mRNAs.Other miRNAs have less than perfect complementarity to their targetmRNAs and, hence, direct translational repression of the target mRNAs.The degree of complementarity between an miRNA and its target mRNA isbelieved to determine its mechanism of action. For example, perfect ornear-perfect complementarity between a miRNA and its target mRNA ispredictive of a cleavage mechanism (Yekta et al., Science, 2004),whereas less than perfect complementarity is predictive of atranslational repression mechanism. In particular embodiments, the miRNAsequence is that of a naturally-occurring miRNA sequence, the aberrantexpression or activity of which is correlated with an miRNA disorder.

d) Dual Functional Oligonucleotide Tethers

In other embodiments, the RNA silencing agents of the present inventioninclude dual functional oligonucleotide tethers useful for theintercellular recruitment of a miRNA. Animal cells express a range ofmiRNAs, noncoding RNAs of approximately 22 nucleotides which canregulate gene expression at the post transcriptional or translationallevel. By binding a miRNA bound to RISC and recruiting it to a targetmRNA, a dual functional oligonucleotide tether can repress theexpression of genes involved e.g., in the arteriosclerotic process. Theuse of oligonucleotide tethers offer several advantages over existingtechniques to repress the expression of a particular gene. First, themethods described herein allow an endogenous molecule (often present inabundance), an miRNA, to mediate RNA silencing. Accordingly, the methodsdescribed herein obviate the need to introduce foreign molecules (e.g.,siRNAs) to mediate RNA silencing. Second, the RNA-silencing agents and,in particular, the linking moiety (e.g., oligonucleotides such as the2′-O-methyl oligonucleotide), can be made stable and resistant tonuclease activity. As a result, the tethers of the present invention canbe designed for direct delivery, obviating the need for indirectdelivery (e.g. viral) of a precursor molecule or plasmid designed tomake the desired agent within the cell. Third, tethers and theirrespective moieties, can be designed to conform to specific mRNA sitesand specific miRNAs. The designs can be cell and gene product specific.Fourth, the methods disclosed herein leave the mRNA intact, allowing oneskilled in the art to block protein synthesis in short pulses using thecell's own machinery. As a result, these methods of RNA silencing arehighly regulatable.

The dual functional oligonucleotide tethers (“tethers”) of the inventionare designed such that they recruit miRNAs (e.g., endogenous cellularmiRNAs) to a target mRNA so as to induce the modulation of a gene ofinterest. In preferred embodiments, the tethers have the formula T-L-μ,wherein T is an mRNA targeting moiety, L is a linking moiety, and μ isan miRNA recruiting moiety. Any one or more moiety may be doublestranded. Preferably, however, each moiety is single stranded.

Moieties within the tethers can be arranged or linked (in the 5′ to 3′direction) as depicted in the formula T-L-μ (i.e., the 3′ end of thetargeting moiety linked to the 5′ end of the linking moiety and the 3′end of the linking moiety linked to the 5′ end of the miRNA recruitingmoiety). Alternatively, the moieties can be arranged or linked in thetether as follows: μ-T-L (i.e., the 3′ end of the miRNA recruitingmoiety linked to the 5′ end of the linking moiety and the 3′ end of thelinking moiety linked to the 5′ end of the targeting moiety).

The mRNA targeting moiety, as described above, is capable of capturing aspecific target mRNA. According to the invention, expression of thetarget mRNA is undesirable, and, thus, translational repression of themRNA is desired. The mRNA targeting moiety should be of sufficient sizeto effectively bind the target mRNA. The length of the targeting moietywill vary greatly depending, in part, on the length of the target mRNAand the degree of complementarity between the target mRNA and thetargeting moiety. In various embodiments, the targeting moiety is lessthan about 200, 100, 50, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, or 5 nucleotides in length. In a particular embodiment,the targeting moiety is about 15 to about 25 nucleotides in length.

The miRNA recruiting moiety, as described above, is capable ofassociating with a miRNA. According to the invention, the miRNA may beany miRNA capable of repressing the target mRNA. Mammals are reported tohave over 250 endogenous miRNAs (Lagos-Quintana et al. (2002) CurrentBiol. 12:735-739; Lagos-Quintana et al. (2001) Science 294:858-862; andLim et al. (2003) Science 299:1540). In various embodiments, the miRNAmay be any art-recognized miRNA.

The linking moiety is any agent capable of linking the targetingmoieties such that the activity of the targeting moieties is maintained.Linking moieties are preferably oligonucleotide moieties comprising asufficient number of nucleotides such that the targeting agents cansufficiently interact with their respective targets. Linking moietieshave little or no sequence homology with cellular mRNA or miRNAsequences. Exemplary linking moieties include one or more2′-O-methylnucleotides, e.g., 2′-β-methyladenosine,2′-O-methylthymidine, 2′-O-methylguanosine or 2′-O-methyluridine.

e) Gene Silencing Oligonucleotides

In certain exemplary embodiments, gene expression (i.e., htt geneexpression) can be modulated using oligonucleotide-based compoundscomprising two or more single stranded antisense oligonucleotides thatare linked through their 5′-ends that allow the presence of two or moreaccessible 3′-ends to effectively inhibit or decrease htt geneexpression. Such linked oligonucleotides are also known as GeneSilencing Oligonucleotides (GSOs). (See, e.g., U.S. Pat. No. 8,431,544assigned to Idera Pharmaceuticals, Inc., incorporated herein byreference in its entirety for all purposes.)

The linkage at the 5′ ends of the GSOs is independent of the otheroligonucleotide linkages and may be directly via 5′, 3′ or 2′ hydroxylgroups, or indirectly, via a non-nucleotide linker or a nucleoside,utilizing either the 2′ or 3′ hydroxyl positions of the nucleoside.Linkages may also utilize a functionalized sugar or nucleobase of a 5′terminal nucleotide.

GSOs can comprise two identical or different sequences conjugated attheir 5′-5′ ends via a phosphodiester, phosphorothioate ornon-nucleoside linker. Such compounds may comprise 15 to 27 nucleotidesthat are complementary to specific portions of mRNA targets of interestfor antisense down regulation of gene product. GSOs that compriseidentical sequences can bind to a specific mRNA via Watson-Crickhydrogen bonding interactions and inhibit protein expression. GSOs thatcomprise different sequences are able to bind to two or more differentregions of one or more mRNA target and inhibit protein expression. Suchcompounds are comprised of heteronucleotide sequences complementary totarget mRNA and form stable duplex structures through Watson-Crickhydrogen bonding. Under certain conditions, GSOs containing two free3′-ends (5′-5′-attached antisense) can be more potent inhibitors of geneexpression than those containing a single free 3′-end or no free 3′-end.

In some embodiments, the non-nucleotide linker is glycerol or a glycerolhomolog of the formula HO—(CH₂)_(o)—CH(OH)—(CH₂)_(p)—OH, wherein o and pindependently are integers from 1 to about 6, from 1 to about 4 or from1 to about 3. In some other embodiments, the non-nucleotide linker is aderivative of 1,3-diamino-2-hydroxypropane. Some such derivatives havethe formula HO—(CH₂)_(m)—C(O)NH—CH₂—CH(OH)—CH₂—NHC(O)—(CH₂)_(m)—OH,wherein m is an integer from 0 to about 10, from 0 to about 6, from 2 toabout 6 or from 2 to about 4.

Some non-nucleotide linkers permit attachment of more than two GSOcomponents. For example, the non-nucleotide linker glycerol has threehydroxyl groups to which GSO components may be covalently attached. Someoligonucleotide-based compounds of the invention, therefore, comprisetwo or more oligonucleotides linked to a nucleotide or a non-nucleotidelinker. Such oligonucleotides according to the invention are referred toas being “branched.”

In certain embodiments, GSOs are at least 14 nucleotides in length. Incertain exemplary embodiments, GSOs are 15 to 40 nucleotides long or 20to 30 nucleotides in length. Thus, the component oligonucleotides ofGSOs can independently be 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40nucleotides in length.

These oligonucleotides can be prepared by the art recognized methodssuch as phosphoramidate or H-phosphonate chemistry which can be carriedout manually or by an automated synthesizer. These oligonucleotides mayalso be modified in a number of ways without compromising their abilityto hybridize to mRNA. Such modifications may include at least oneinternucleotide linkage of the oligonucleotide being analkylphosphonate, phosphorothioate, phosphorodithioate,methylphosphonate, phosphate ester, alkylphosphonothioate,phosphoramidate, carbamate, carbonate, phosphate hydroxyl, acetamidateor carboxymethyl ester or a combination of these and otherinternucleotide linkages between the 5′ end of one nucleotide and the 3′end of another nucleotide in which the 5′ nucleotide phosphodiesterlinkage has been replaced with any number of chemical groups.

VII. Modified Anti-Htt RNA Silencing Agents

In certain aspects of the invention, an RNA silencing agent (or anyportion thereof) of the invention as described supra may be modifiedsuch that the activity of the agent is further improved. For example,the RNA silencing agents described in Section II supra may be modifiedwith any of the modifications described infra. The modifications can, inpart, serve to further enhance target discrimination, to enhancestability of the agent (e.g., to prevent degradation), to promotecellular uptake, to enhance the target efficiency, to improve efficacyin binding (e.g., to the targets), to improve patient tolerance to theagent, and/or to reduce toxicity.

1) Modifications to Enhance Target Discrimination

In certain embodiments, the RNA silencing agents of the invention may besubstituted with a destabilizing nucleotide to enhance single nucleotidetarget discrimination (see U.S. application Ser. No. 11/698,689, filedJan. 25, 2007 and U.S. Provisional Application No. 60/762,225 filed Jan.25, 2006, both of which are incorporated herein by reference). Such amodification may be sufficient to abolish the specificity of the RNAsilencing agent for a non-target mRNA (e.g. wild-type mRNA), withoutappreciably affecting the specificity of the RNA silencing agent for atarget mRNA (e.g. gain-of-function mutant mRNA).

In preferred embodiments, the RNA silencing agents of the invention aremodified by the introduction of at least one universal nucleotide in theantisense strand thereof. Universal nucleotides comprise base portionsthat are capable of base pairing indiscriminately with any of the fourconventional nucleotide bases (e.g. A, G, C, U). A universal nucleotideis preferred because it has relatively minor effect on the stability ofthe RNA duplex or the duplex formed by the guide strand of the RNAsilencing agent and the target mRNA. Exemplary universal nucleotideinclude those having an inosine base portion or an inosine analog baseportion selected from the group consisting of deoxyinosine (e.g.2′-deoxyinosine), 7-deaza-2′-deoxyinosine, 2′-aza-2′-deoxyinosine,PNA-inosine, morpholino-inosine, LNA-inosine, phosphoramidate-inosine,2′-O-methoxyethyl-inosine, and 2′-OMe-inosine. In particularly preferredembodiments, the universal nucleotide is an inosine residue or anaturally occurring analog thereof.

In certain embodiments, the RNA silencing agents of the invention aremodified by the introduction of at least one destabilizing nucleotidewithin 5 nucleotides from a specificity-determining nucleotide (i.e.,the nucleotide which recognizes the disease-related polymorphism). Forexample, the destabilizing nucleotide may be introduced at a positionthat is within 5, 4, 3, 2, or 1 nucleotide(s) from aspecificity-determining nucleotide. In exemplary embodiments, thedestabilizing nucleotide is introduced at a position which is 3nucleotides from the specificity-determining nucleotide (i.e., such thatthere are 2 stabilizing nucleotides between the destablilizingnucleotide and the specificity-determining nucleotide). In RNA silencingagents having two strands or strand portions (e.g. siRNAs and shRNAs),the destabilizing nucleotide may be introduced in the strand or strandportion that does not contain the specificity-determining nucleotide. Inpreferred embodiments, the destabilizing nucleotide is introduced in thesame strand or strand portion that contains the specificity-determiningnucleotide.

2) Modifications to Enhance Efficacy and Specificity

In certain embodiments, the RNA silencing agents of the invention may bealtered to facilitate enhanced efficacy and specificity in mediatingRNAi according to asymmetry design rules (see U.S. Pat. Nos. 8,309,704,7,750,144, 8,304,530, 8,329,892 and 8,309,705). Such alterationsfacilitate entry of the antisense strand of the siRNA (e.g., a siRNAdesigned using the methods of the invention or an siRNA produced from ashRNA) into RISC in favor of the sense strand, such that the antisensestrand preferentially guides cleavage or translational repression of atarget mRNA, and thus increasing or improving the efficiency of targetcleavage and silencing. Preferably the asymmetry of an RNA silencingagent is enhanced by lessening the base pair strength between theantisense strand 5′ end (AS 5′) and the sense strand 3′ end (S 3′) ofthe RNA silencing agent relative to the bond strength or base pairstrength between the antisense strand 3′ end (AS 3′) and the sensestrand 5′ end (S ′5) of said RNA silencing agent.

In one embodiment, the asymmetry of an RNA silencing agent of theinvention may be enhanced such that there are fewer G:C base pairsbetween the 5′ end of the first or antisense strand and the 3′ end ofthe sense strand portion than between the 3′ end of the first orantisense strand and the 5′ end of the sense strand portion. In anotherembodiment, the asymmetry of an RNA silencing agent of the invention maybe enhanced such that there is at least one mismatched base pair betweenthe 5′ end of the first or antisense strand and the 3′ end of the sensestrand portion. Preferably, the mismatched base pair is selected fromthe group consisting of G:A, C:A, C:U, G:G, A:A, C:C and U:U. In anotherembodiment, the asymmetry of an RNA silencing agent of the invention maybe enhanced such that there is at least one wobble base pair, e.g., G:U,between the 5′ end of the first or antisense strand and the 3′ end ofthe sense strand portion. In another embodiment, the asymmetry of an RNAsilencing agent of the invention may be enhanced such that there is atleast one base pair comprising a rare nucleotide, e.g., inosine (I).Preferably, the base pair is selected from the group consisting of anI:A, I:U and I:C. In yet another embodiment, the asymmetry of an RNAsilencing agent of the invention may be enhanced such that there is atleast one base pair comprising a modified nucleotide. In preferredembodiments, the modified nucleotide is selected from the groupconsisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

3) RNA Silencing Agents with Enhanced Stability

The RNA silencing agents of the present invention can be modified toimprove stability in serum or in growth medium for cell cultures. Inorder to enhance the stability, the 3′-residues may be stabilizedagainst degradation, e.g., they may be selected such that they consistof purine nucleotides, particularly adenosine or guanosine nucleotides.Alternatively, substitution of pyrimidine nucleotides by modifiedanalogues, e.g., substitution of uridine by 2′-deoxythymidine istolerated and does not affect the efficiency of RNA interference.

In a preferred aspect, the invention features RNA silencing agents thatinclude first and second strands wherein the second strand and/or firststrand is modified by the substitution of internal nucleotides withmodified nucleotides, such that in vivo stability is enhanced ascompared to a corresponding unmodified RNA silencing agent. As definedherein, an “internal” nucleotide is one occurring at any position otherthan the 5′ end or 3′ end of nucleic acid molecule, polynucleotide oroligonucleotide. An internal nucleotide can be within a single-strandedmolecule or within a strand of a duplex or double-stranded molecule. Inone embodiment, the sense strand and/or antisense strand is modified bythe substitution of at least one internal nucleotide. In anotherembodiment, the sense strand and/or antisense strand is modified by thesubstitution of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more internal nucleotides. Inanother embodiment, the sense strand and/or antisense strand is modifiedby the substitution of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of theinternal nucleotides. In yet another embodiment, the sense strand and/orantisense strand is modified by the substitution of all of the internalnucleotides.

In a preferred embodiment of the present invention, the RNA silencingagents may contain at least one modified nucleotide analogue. Thenucleotide analogues may be located at positions where thetarget-specific silencing activity, e.g., the RNAi mediating activity ortranslational repression activity is not substantially effected, e.g.,in a region at the 5′-end and/or the 3′-end of the siRNA molecule.Particularly, the ends may be stabilized by incorporating modifiednucleotide analogues.

Exemplary nucleotide analogues include sugar- and/or backbone-modifiedribonucleotides (i.e., include modifications to the phosphate-sugarbackbone). For example, the phosphodiester linkages of natural RNA maybe modified to include at least one of a nitrogen or sulfur heteroatom.In exemplary backbone-modified ribonucleotides, the phosphoester groupconnecting to adjacent ribonucleotides is replaced by a modified group,e.g., of phosphothioate group. In exemplary sugar-modifiedribonucleotides, the 2′ OH-group is replaced by a group selected from H,OR, R, halo, SH, SR, NH₂, NHR, NR₂ or ON, wherein R is C₁-C₆ alkyl,alkenyl or alkynyl and halo is F, Cl, Br or I.

In particular embodiments, the modifications are 2′-fluoro, 2′-aminoand/or 2′-thio modifications. Particularly preferred modificationsinclude 2′-fluoro-cytidine, 2′-fluoro-uridine, 2′-fluoro-adenosine,2′-fluoro-guanosine, 2′-amino-cytidine, 2′-amino-uridine,2′-amino-adenosine, 2′-amino-guanosine, 2,6-diaminopurine,4-thio-uridine, and/or 5-amino-allyl-uridine. In a particularembodiment, the 2′-fluoro ribonucleotides are every uridine andcytidine. Additional exemplary modifications include 5-bromo-uridine,5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine,2′-amino-butyryl-pyrene-uridine, 5-fluoro-cytidine, and5-fluoro-uridine. 2′-deoxy-nucleotides and 2′-Ome nucleotides can alsobe used within modified RNA-silencing agents moities of the instantinvention. Additional modified residues include, deoxy-abasic, inosine,N3-methyl-uridine, N6,N6-dimethyl-adenosine, pseudouridine, purineribonucleoside and ribavirin. In a particularly preferred embodiment,the 2′ moiety is a methyl group such that the linking moiety is a2′-O-methyl oligonucleotide.

In an exemplary embodiment, the RNA silencing agent of the inventioncomprises Locked Nucleic Acids (LNAs). LNAs comprise sugar-modifiednucleotides that resist nuclease activities (are highly stable) andpossess single nucleotide discrimination for mRNA (Elmen et al., NucleicAcids Res., (2005), 33(1): 439-447; Braasch et al. (2003) Biochemistry42:7967-7975, Petersen et al. (2003) Trends Biotechnol 21:74-81). Thesemolecules have 2′-O,4′-C-ethylene-bridged nucleic acids, with possiblemodifications such as 2′-deoxy-2″-fluorouridine. Moreover, LNAs increasethe specificity of oligonucleotides by constraining the sugar moietyinto the 3′-endo conformation, thereby pre-organizing the nucleotide forbase pairing and increasing the melting temperature of theoligonucleotide by as much as 10° C. per base.

In another exemplary embodiment, the RNA silencing agent of theinvention comprises Peptide Nucleic Acids (PNAs). PNAs comprise modifiednucleotides in which the sugar-phosphate portion of the nucleotide isreplaced with a neutral 2-amino ethylglycine moiety capable of forming apolyamide backbone which is highly resistant to nuclease digestion andimparts improved binding specificity to the molecule (Nielsen, et al.,Science, (2001), 254: 1497-1500).

Also preferred are nucleobase-modified ribonucleotides, i.e.,ribonucleotides, containing at least one non-naturally occurringnucleobase instead of a naturally occurring nucleobase. Bases may bemodified to block the activity of adenosine deaminase. Exemplarymodified nucleobases include, but are not limited to, uridine and/orcytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine,5-bromo uridine; adenosine and/or guanosines modified at the 8 position,e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O-and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. Itshould be noted that the above modifications may be combined.

In other embodiments, cross-linking can be employed to alter thepharmacokinetics of the RNA silencing agent, for example, to increasehalf-life in the body. Thus, the invention includes RNA silencing agentshaving two complementary strands of nucleic acid, wherein the twostrands are crosslinked. The invention also includes RNA silencingagents which are conjugated or unconjugated (e.g., at its 3′ terminus)to another moiety (e.g. a non-nucleic acid moiety such as a peptide), anorganic compound (e.g., a dye), or the like). Modifying siRNAderivatives in this way may improve cellular uptake or enhance cellulartargeting activities of the resulting siRNA derivative as compared tothe corresponding siRNA, are useful for tracing the siRNA derivative inthe cell, or improve the stability of the siRNA derivative compared tothe corresponding siRNA.

Other exemplary modifications include: (a) 2′ modification, e.g.,provision of a 2′ OMe moiety on a U in a sense or antisense strand, butespecially on a sense strand, or provision of a 2′ OMe moiety in a 3′overhang, e.g., at the 3′ terminus (3′ terminus means at the 3′ atom ofthe molecule or at the most 3′ moiety, e.g., the most 3′ P or 2′position, as indicated by the context); (b) modification of thebackbone, e.g., with the replacement of an 0 with an S, in the phosphatebackbone, e.g., the provision of a phosphorothioate modification, on theU or the A or both, especially on an antisense strand; e.g., with thereplacement of a O with an S; (c) replacement of the U with a C5 aminolinker; (d) replacement of an A with a G (sequence changes are preferredto be located on the sense strand and not the antisense strand); and (d)modification at the 2′, 6′, 7′, or 8′ position. Exemplary embodimentsare those in which one or more of these modifications are present on thesense but not the antisense strand, or embodiments where the antisensestrand has fewer of such modifications. Yet other exemplarymodifications include the use of a methylated P in a 3′ overhang, e.g.,at the 3′ terminus; combination of a 2′ modification, e.g., provision ofa 2′ O Me moiety and modification of the backbone, e.g., with thereplacement of a O with an S, e.g., the provision of a phosphorothioatemodification, or the use of a methylated P, in a 3′ overhang, e.g., atthe 3′ terminus; modification with a 3′ alkyl; modification with anabasic pyrrolidone in a 3′ overhang, e.g., at the 3′ terminus;modification with naproxen, ibuprofen, or other moieties which inhibitdegradation at the 3′ terminus.

4) Modifications to Enhance Cellular Uptake

In other embodiments, RNA silencing agents may be modified with chemicalmoieties, for example, to enhance cellular uptake by target cells (e.g.,neuronal cells). Thus, the invention includes RNA silencing agents whichare conjugated or unconjugated (e.g., at its 3′ terminus) to anothermoiety (e.g. a non-nucleic acid moiety such as a peptide), an organiccompound (e.g., a dye), or the like. The conjugation can be accomplishedby methods known in the art, e.g., using the methods of Lambert et al.,Drug Deliv. Rev.: 47(1), 99-112 (2001) (describes nucleic acids loadedto polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al., J.Control Release 53(1-3):137-43 (1998) (describes nucleic acids bound tonanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4:55-8 (1994)(describes nucleic acids linked to intercalating agents, hydrophobicgroups, polycations or PACA nanoparticles); and Godard et al., Eur. J.Biochem. 232(2):404-10 (1995) (describes nucleic acids linked tonanoparticles).

In a particular embodiment, an RNA silencing agent of invention isconjugated to a lipophilic moiety. In one embodiment, the lipophilicmoiety is a ligand that includes a cationic group. In anotherembodiment, the lipophilic moiety is attached to one or both strands ofan siRNA. In an exemplary embodiment, the lipophilic moiety is attachedto one end of the sense strand of the siRNA. In another exemplaryembodiment, the lipophilic moiety is attached to the 3′ end of the sensestrand. In certain embodiments, the lipophilic moiety is selected fromthe group consisting of cholesterol, vitamin E, vitamin K, vitamin A,folic acid, or a cationic dye (e.g., Cy3). In an exemplary embodiment,the lipophilic moiety is a cholesterol. Other lipophilic moietiesinclude cholic acid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

5) Tethered Ligands

Other entities can be tethered to an RNA silencing agent of theinvention. For example, a ligand tethered to an RNA silencing agent toimprove stability, hybridization thermodynamics with a target nucleicacid, targeting to a particular tissue or cell-type, or cellpermeability, e.g., by an endocytosis-dependent or -independentmechanism. Ligands and associated modifications can also increasesequence specificity and consequently decrease off-site targeting. Atethered ligand can include one or more modified bases or sugars thatcan function as intercalators. These are preferably located in aninternal region, such as in a bulge of RNA silencing agent/targetduplex. The intercalator can be an aromatic, e.g., a polycyclic aromaticor heterocyclic aromatic compound. A polycyclic intercalator can havestacking capabilities, and can include systems with 2, 3, or 4 fusedrings. The universal bases described herein can be included on a ligand.In one embodiment, the ligand can include a cleaving group thatcontributes to target gene inhibition by cleavage of the target nucleicacid. The cleaving group can be, for example, a bleomycin (e.g.,bleomycin-A5, bleomycin-A2, or bleomycin-B2), pyrene, phenanthroline(e.g., O-phenanthroline), a polyamine, a tripeptide (e.g., lys-tyr-lystripeptide), or metal ion chelating group. The metal ion chelating groupcan include, e.g., an Lu(III) or EU(III) macrocyclic complex, a Zn(II)2,9-dimethylphenanthroline derivative, a Cu(II) terpyridine, oracridine, which can promote the selective cleavage of target RNA at thesite of the bulge by free metal ions, such as Lu(III). In someembodiments, a peptide ligand can be tethered to a RNA silencing agentto promote cleavage of the target RNA, e.g., at the bulge region. Forexample, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (cyclam) canbe conjugated to a peptide (e.g., by an amino acid derivative) topromote target RNA cleavage. A tethered ligand can be an aminoglycosideligand, which can cause an RNA silencing agent to have improvedhybridization properties or improved sequence specificity. Exemplaryaminoglycosides include glycosylated polylysine, galactosylatedpolylysine, neomycin B, tobramycin, kanamycin A, and acridine conjugatesof aminoglycosides, such as Neo-N-acridine, Neo-S-acridine,Neo-C-acridine, Tobra-N-acridine, and KanaA-N-acridine. Use of anacridine analog can increase sequence specificity. For example, neomycinB has a high affinity for RNA as compared to DNA, but lowsequence-specificity. An acridine analog, neo-5-acridine has anincreased affinity for the HIV Rev-response element (RRE). In someembodiments the guanidine analog (the guanidinoglycoside) of anaminoglycoside ligand is tethered to an RNA silencing agent. In aguanidinoglycoside, the amine group on the amino acid is exchanged for aguanidine group. Attachment of a guanidine analog can enhance cellpermeability of an RNA silencing agent. A tethered ligand can be apoly-arginine peptide, peptoid or peptidomimetic, which can enhance thecellular uptake of an oligonucleotide agent.

Exemplary ligands are coupled, preferably covalently, either directly orindirectly via an intervening tether, to a ligand-conjugated carrier. Inexemplary embodiments, the ligand is attached to the carrier via anintervening tether. In exemplary embodiments, a ligand alters thedistribution, targeting or lifetime of an RNA silencing agent into whichit is incorporated. In exemplary embodiments, a ligand provides anenhanced affinity for a selected target, e.g., molecule, cell or celltype, compartment, e.g., a cellular or organ compartment, tissue, organor region of the body, as, e.g., compared to a species absent such aligand.

Exemplary ligands can improve transport, hybridization, and specificityproperties and may also improve nuclease resistance of the resultantnatural or modified RNA silencing agent, or a polymeric moleculecomprising any combination of monomers described herein and/or naturalor modified ribonucleotides. Ligands in general can include therapeuticmodifiers, e.g., for enhancing uptake; diagnostic compounds or reportergroups e.g., for monitoring distribution; cross-linking agents;nuclease-resistance conferring moieties; and natural or unusualnucleobases. General examples include lipophiles, lipids, steroids(e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g.,sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid),vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal),carbohydrates, proteins, protein binding agents, integrin targetingmolecules, polycationics, peptides, polyamines, and peptide mimics.Ligands can include a naturally occurring substance, (e.g., human serumalbumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate(e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin orhyaluronic acid); amino acid, or a lipid. The ligand may also be arecombinant or synthetic molecule, such as a synthetic polymer, e.g., asynthetic polyamino acid. Examples of polyamino acids include polyaminoacid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied)copolymer, divinyl ether-maleic anhydride copolymer,N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol(PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllicacid), N-isopropylacrylamide polymers, or polyphosphazine. Example ofpolyamines include: polyethylenimine, polylysine (PLL), spermine,spermidine, polyamine, pseudopeptide-polyamine, peptidomimeticpolyamine, dendrimer polyamine, arginine, amidine, protamine, cationiclipid, cationic porphyrin, quaternary salt of a polyamine, or an alphahelical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine, multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide orRGD peptide mimetic. Other examples of ligands include dyes,intercalating agents (e.g. acridines and substituted acridines),cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4,texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine, phenanthroline, pyrenes), lys-tyr-lystripeptide, aminoglycosides, guanidium aminoglycodies, artificialendonucleases (e.g. EDTA), lipophilic molecules, e.g, cholesterol (andthio analogs thereof), cholic acid, cholanic acid, lithocholic acid,adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,glycerol (e.g., esters (e.g., mono, bis, or tris fatty acid esters,e.g., C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, or C₂₀ fattyacids) and ethers thereof, e.g., C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇,C₁₈, C₁₉, or C₂₀ alkyl; e.g., 1,3-bis-O(hexadecyl)glycerol,1,3-bis-O(octaadecyl)glycerol), geranyloxyhexyl group,hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group,palmitic acid, stearic acid (e.g., glyceryl distearate), oleic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, naproxen, vitamin E,folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, or multivalent fucose. Theligand can be, for example, a lipopolysaccharide, an activator of p38MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the RNA silencing agent into the cell, for example, bydisrupting the cell's cytoskeleton, e.g., by disrupting the cell'smicrotubules, microfilaments, and/or intermediate filaments. The drugcan be, for example, taxon, vincristine, vinblastine, cytochalasin,nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A,indanocine, or myoservin. The ligand can increase the uptake of the RNAsilencing agent into the cell by activating an inflammatory response,for example. Exemplary ligands that would have such an effect includetumor necrosis factor alpha (TNFα), interleukin-1 beta, or gammainterferon. In one aspect, the ligand is a lipid or lipid-basedmolecule. Such a lipid or lipid-based molecule preferably binds a serumprotein, e.g., human serum albumin (HSA). An HSA binding ligand allowsfor distribution of the conjugate to a target tissue, e.g., a non-kidneytarget tissue of the body. For example, the target tissue can be theliver, including parenchymal cells of the liver. Other molecules thatcan bind HSA can also be used as ligands. For example, neproxin oraspirin can be used. A lipid or lipid-based ligand can (a) increaseresistance to degradation of the conjugate, (b) increase targeting ortransport into a target cell or cell membrane, and/or (c) can be used toadjust binding to a serum protein, e.g., HSA. A lipid based ligand canbe used to modulate, e.g., control the binding of the conjugate to atarget tissue. For example, a lipid or lipid-based ligand that binds toHSA more strongly will be less likely to be targeted to the kidney andtherefore less likely to be cleared from the body. A lipid orlipid-based ligand that binds to HSA less strongly can be used to targetthe conjugate to the kidney. In a preferred embodiment, the lipid basedligand binds HSA. A lipid-based ligand can bind HSA with a sufficientaffinity such that the conjugate will be preferably distributed to anon-kidney tissue. However, it is preferred that the affinity not be sostrong that the HSA-ligand binding cannot be reversed. In anotherpreferred embodiment, the lipid based ligand binds HSA weakly or not atall, such that the conjugate will be preferably distributed to thekidney. Other moieties that target to kidney cells can also be used inplace of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HSA and low density lipoprotein (LDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics tooligonucleotide agents can affect pharmacokinetic distribution of theRNA silencing agent, such as by enhancing cellular recognition andabsorption. The peptide or peptidomimetic moiety can be about 5-50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long. A peptide or peptidomimetic can be, for example, a cellpermeation peptide, cationic peptide, amphipathic peptide, orhydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). Thepeptide moiety can be a dendrimer peptide, constrained peptide orcrosslinked peptide. The peptide moiety can be an L-peptide orD-peptide. In another alternative, the peptide moiety can include ahydrophobic membrane translocation sequence (MTS). A peptide orpeptidomimetic can be encoded by a random sequence of DNA, such as apeptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature354:82-84, 1991). In exemplary embodiments, the peptide orpeptidomimetic tethered to an RNA silencing agent via an incorporatedmonomer unit is a cell targeting peptide such as anarginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptidemoiety can range in length from about 5 amino acids to about 40 aminoacids. The peptide moieties can have a structural modification, such asto increase stability or direct conformational properties. Any of thestructural modifications described below can be utilized.

6) Compounds

In one aspect, provided herein is a compound of the Formula shown inFIG. 81, or a pharmaceutically acceptable salt thereof, wherein

-   R¹ is selected from the group consisting of:

-   R³ is independently selected at each occurrence from the group    consisting of an internucleotide linker as shown in FIG. 82; and-   L is a linker connecting two moieties, wherein the linker is    selected from the group consisting of an ethylene glycol chain, an    alkyl chain, a peptide, an RNA, a DNA,

-   or a combination thereof.

In one embodiment, R¹ is selected from the group consisting of

In another embodiment, R¹ is

In another embodiment, R³ is an internucleotide linker independentlyselected at each occurrence from the group consisting of aphosphorothioate, a phosphorodithioate, a methylphosphonate, amethylenephosphonate, a phosphotriester, and a boranophosphate.

In another embodiment, R³ is an internucleotide linker independentlyselected at each occurrence from the group consisting of aphosphorothioate, a phosphorodithioate, and a boranophosphate.

In another embodiment, R³ is a phosphorothioate.

In another embodiment, L is selected from the group consisting of anethylene glycol chain, an alkyl chain, and a peptide.

In another embodiment, L is selected from an ethylene glycol chain or apeptide.

In yet another embodiment, L is

In still another embodiment, L is

In another embodiment, L is

In one embodiment, the compound of the Formula shown in FIG. 81 is acompound the Formula shown in FIG. 83.

In another embodiment, the compound of the Formula shown in FIG. 81 is acompound of the Formula shown in FIG. 83, or a pharmaceuticallyacceptable salt thereof, wherein

-   R¹ is

and

-   L is

In another embodiment, the compound of the Formula shown in FIG. 81 is acompound of the Formula shown in FIG. 83, or a pharmaceuticallyacceptable salt thereof, wherein

-   R¹ is

and

-   L is

In another aspect, provided herein is a compound of the Formula shown inFIG. 84, or a pharmaceutically acceptable salt thereof, wherein

-   R¹ is selected from the group consisting of

-   R³ is independently selected at each occurrence from the group    consisting of an internucleotide linker as shown in FIG. 82;-   L is a linker connecting two moieties, wherein the linker is    selected from the group consisting of an ethylene glycol chain, an    alkyl chain, a peptide, an RNA, a DNA,

-   or a combination thereof; and-   B is a branch point between two or more linkers, wherein the branch    point is selected from the group consisting of a glycol, an amino    acid, or any poly-valent organic species.

In one embodiment, R¹ is selected from the group consisting of

In another embodiment, R¹ is

In another embodiment, R³ is an internucleotide linker independentlyselected at each occurrence from the group consisting of aphosphorothioate, a phosphorodithioate, a methylphosphonate, amethylenephosphonate, a phosphotriester, and a boranophosphate.

In another embodiment, R³ is an internucleotide linker independentlyselected at each occurrence from the group consisting of aphosphorothioate, a phosphorodithioate, and a boranophosphate.

In another embodiment, R³ is a phosphorothioate.

In another embodiment, L is selected from the group consisting of anethylene glycol chain, an alkyl chain, and a peptide.

In another embodiment, L is selected from an ethylene glycol chain or apeptide.

In yet another embodiment, L is

In still another embodiment, L is

In another embodiment, L is

In one embodiment, B is a branch point between two or more linkers,wherein the branch point is selected a glycol or an amino acid. Inanother embodiment, the branch point is a glycol. In another embodiment,the branch point is an amino acid.

In another embodiment of the compound of the Formula shown in FIG. 84, Yis defined as shown in FIG. 86.

In another aspect, provided herein is a compound of the Formula shown inFIG. 85, or a pharmaceutically acceptable salt thereof, wherein

R¹ is selected from the group consisting of

-   R³ is independently selected at each occurrence from the group    consisting of an internucleotide linker as shown in FIG. 82;-   L is a linker connecting two moieties, wherein the linker is    selected from the group consisting of an ethylene glycol chain, an    alkyl chain, a peptide, an RNA, a DNA,

-   or a combination thereof; and-   B is a branch point between two or more linkers, wherein the branch    point is selected from the group consisting of a glycol, an amino    acid, or any polyvalent organic species.

In one embodiment, R¹ is selected from the group consisting of

In another embodiment, R¹ is

In another embodiment, R³ is an internucleotide linker independentlyselected at each occurrence from the group consisting of aphosphorothioate, a phosphorodithioate, a methylphosphonate, amethylenephosphonate, a phosphotriester, and a boranophosphate.

In another embodiment, R³ is an internucleotide linker independentlyselected at each occurrence from the group consisting of aphosphorothioate, a phosphorodithioate, and a boranophosphate.

In another embodiment, R³ is a phosphorothioate.

In another embodiment, L is selected from the group consisting of anethylene glycol chain, an alkyl chain, and a peptide.

In another embodiment, L is selected from an ethylene glycol chain or apeptide.

In yet another embodiment, L is

In still another embodiment, L is

In another embodiment, L is

In one embodiment, B is a branch point between two or more linkers,wherein the branch point is selected a glycol or an amino acid. Inanother embodiment, the branch point is a glycol. In another embodiment,the branch point is an amino acid.

In one embodiment of the compound of the Formula shown in FIG. 85, Y isdefined as shown in FIG. 86.

In another aspect, provided herein is a compound of the Formula shown inFIG. 87, or a pharmaceutically acceptable salt thereof, wherein

-   R¹ is selected from the group consisting of

-   R³ is independently selected at each occurrence from the group    consisting of an internucleotide linker as shown in FIG. 82;-   L is a linker connecting two moieties, wherein the linker is    selected from the group consisting of an ethylene glycol chain, an    alkyl chain, a peptide, an RNA, a DNA,

-   or a combination thereof; and-   R² is selected from the group consisting of an alkyl chain (e.g.,    C₁₋₆, C₁₋₁₀, C₁₋₂₀, C₁₋₃₀, or C₁₋₄₀), a vitamin, a ligand, a    peptide, a bioactive conjugate (including, but not limited to    glycosphingolipids, polyunsaturated fatty acids, secosteroids,    steroid hormones, or sterol lipids),

In one embodiment, R¹ is selected from the group consisting of

In another embodiment, R¹ is

In another embodiment, R² is selected from the group consisting of

In another embodiment, R³ is an internucleotide linker independentlyselected at each occurrence from the group consisting of aphosphorothioate, a phosphorodithioate, a methylphosphonate, amethylenephosphonate, a phosphotriester, and a boranophosphate.

In another embodiment, R³ is an internucleotide linker independentlyselected at each occurrence from the group consisting of aphosphorothioate, a phosphorodithioate, and a boranophosphate.

In another embodiment, R³ is a phosphorothioate.

In another embodiment, L is selected from the group consisting of anethylene glycol chain, an alkyl chain, and a peptide.

In another embodiment, L is selected from an ethylene glycol chain or apeptide.

In yet another embodiment, L is

In still another embodiment, L is

In another embodiment, L is

In one embodiment, the compound of the Formula shown in FIG. 87 is acompound the Formula shown in FIG. 88.

In another embodiment, the compound of the Formula shown in FIG. 87, isa compound of the Formula shown in FIG. 88, or a pharmaceuticallyacceptable salt thereof, wherein

-   R¹ is

and

-   R² is

In another aspect, provided herein is a compound of the Formula shown inFIG. 89, or a pharmaceutically acceptable salt thereof, wherein

-   R¹ is selected from the group consisting of

-   R³ is independently selected at each occurrence from the group    consisting of an internucleotide linker as shown in FIG. 82; and-   R² is selected from the group consisting of an alkyl chain (e.g.,    C₁₋₆, C₁₋₁₀, C₁₋₂₀, C₁₋₃₀, or C₁₋₄₀), a vitamin, a ligand, a    peptide, a bioactive conjugate (including, but not limited to    glycosphingolipids, polyunsaturated fatty acids, secosteroids,    steroid hormones, or sterol lipids),

In one embodiment, R¹ is selected from the group consisting of

In another embodiment, R¹ is

In another embodiment, R² is selected from the group consisting of

In another embodiment, R³ is an internucleotide linker independentlyselected at each occurrence from the group consisting of aphosphorothioate, a phosphorodithioate, a methylphosphonate, amethylenephosphonate, a phosphotriester, and a boranophosphate.

In another embodiment, R³ is an internucleotide linker independentlyselected at each occurrence from the group consisting of aphosphorothioate, a phosphorodithioate, and a boranophosphate.

In another embodiment, R³ is a phosphorothioate.

In one embodiment, the compound of the Formula shown in FIG. 89 is acompound of the Formula shown in FIG. 90.

In one embodiment, the compound of the Formula shown in FIG. 89, is acompound of the Formula shown in FIG. 90, or a pharmaceuticallyacceptable salt thereof, wherein

-   R¹ is

and

-   R² is

In one embodiment, the compound of the Formula shown in FIG. 89, is acompound of the Formula shown in FIG. 90, or a pharmaceuticallyacceptable salt thereof, wherein

-   R¹ is

and

-   R² is

In one embodiment, the compound of the Formula shown in FIG. 89, is acompound of the Formula shown in FIG. 90, or a pharmaceuticallyacceptable salt thereof, wherein

-   R¹ is

and

-   R² is

In one embodiment, the compound of the Formula shown in FIG. 89, is acompound of the Formula shown in FIG. 90, or a pharmaceuticallyacceptable salt thereof, wherein

-   R¹ is

and

-   R² is

In one embodiment, the compound of the Formula shown in FIG. 89, is acompound of the Formula shown in FIG. 90, or a pharmaceuticallyacceptable salt thereof, wherein

-   R¹ is

and

-   R² is

In one embodiment, the compound of the Formula shown in FIG. 89, is acompound of the Formula shown in FIG. 90, or a pharmaceuticallyacceptable salt thereof, wherein

-   R¹ is

and

-   R² is

VIII. Methods of Introducing Nucleic Acids, Vectors and Host Cells

RNA silencing agents of the invention may be directly introduced intothe cell (e.g., a neural cell) (i.e., intracellularly); or introducedextracellularly into a cavity, interstitial space, into the circulationof an organism, introduced orally, or may be introduced by bathing acell or organism in a solution containing the nucleic acid. Vascular orextravascular circulation, the blood or lymph system, and thecerebrospinal fluid are sites where the nucleic acid may be introduced.

The RNA silencing agents of the invention can be introduced usingnucleic acid delivery methods known in art including injection of asolution containing the nucleic acid, bombardment by particles coveredby the nucleic acid, soaking the cell or organism in a solution of thenucleic acid, or electroporation of cell membranes in the presence ofthe nucleic acid. Other methods known in the art for introducing nucleicacids to cells may be used, such as lipid-mediated carrier transport,chemical-mediated transport, and cationic liposome transfection such ascalcium phosphate, and the like. The nucleic acid may be introducedalong with other components that perform one or more of the followingactivities: enhance nucleic acid uptake by the cell or other-wiseincrease inhibition of the target gene.

Physical methods of introducing nucleic acids include injection of asolution containing the RNA, bombardment by particles covered by theRNA, soaking the cell or organism in a solution of the RNA, orelectroporation of cell membranes in the presence of the RNA. A viralconstruct packaged into a viral particle would accomplish both efficientintroduction of an expression construct into the cell and transcriptionof RNA encoded by the expression construct. Other methods known in theart for introducing nucleic acids to cells may be used, such aslipid-mediated carrier transport, chemical-mediated transport, such ascalcium phosphate, and the like. Thus the RNA may be introduced alongwith components that perform one or more of the following activities:enhance RNA uptake by the cell, inhibit annealing of single strands,stabilize the single strands, or other-wise increase inhibition of thetarget gene.

RNA may be directly introduced into the cell (i.e., intracellularly); orintroduced extracellularly into a cavity, interstitial space, into thecirculation of an organism, introduced orally, or may be introduced bybathing a cell or organism in a solution containing the RNA. Vascular orextravascular circulation, the blood or lymph system, and thecerebrospinal fluid are sites where the RNA may be introduced.

The cell having the target gene may be from the germ line or somatic,totipotent or pluripotent, dividing or non-dividing, parenchyma orepithelium, immortalized or transformed, or the like. The cell may be astem cell or a differentiated cell. Cell types that are differentiatedinclude adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium,neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages,neutrophils, eosinophils, basophils, mast cells, leukocytes,granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts,hepatocytes, and cells of the endocrine or exocrine glands.

Depending on the particular target gene and the dose of double strandedRNA material delivered, this process may provide partial or completeloss of function for the target gene. A reduction or loss of geneexpression in at least 50%, 60%, 70%, 80%, 90%, 95% or 99% or more oftargeted cells is exemplary. Inhibition of gene expression refers to theabsence (or observable decrease) in the level of protein and/or mRNAproduct from a target gene. Specificity refers to the ability to inhibitthe target gene without manifest effects on other genes of the cell. Theconsequences of inhibition can be confirmed by examination of theoutward properties of the cell or organism (as presented below in theexamples) or by biochemical techniques such as RNA solutionhybridization, nuclease protection, Northern hybridization, reversetranscription, gene expression monitoring with a microarray, antibodybinding, Enzyme Linked ImmunoSorbent Assay (ELISA), Western blotting,RadiolmmunoAssay (RIA), other immunoassays, and Fluorescence ActivatedCell Sorting (FACS).

For RNA-mediated inhibition in a cell line or whole organism, geneexpression is conveniently assayed by use of a reporter or drugresistance gene whose protein product is easily assayed. Such reportergenes include acetohydroxyacid synthase (AHAS), alkaline phosphatase(AP), beta galactosidase (LacZ), beta glucoronidase (GUS),chloramphenicol acetyltransferase (CAT), green fluorescent protein(GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase(NOS), octopine synthase (OCS), and derivatives thereof. Multipleselectable markers are available that confer resistance to ampicillin,bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin,lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin.Depending on the assay, quantitation of the amount of gene expressionallows one to determine a degree of inhibition which is greater than10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treatedaccording to the present invention. Lower doses of injected material andlonger times after administration of RNAi agent may result in inhibitionin a smaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%,or 95% of targeted cells). Quantization of gene expression in a cell mayshow similar amounts of inhibition at the level of accumulation oftarget mRNA or translation of target protein. As an example, theefficiency of inhibition may be determined by assessing the amount ofgene product in the cell; mRNA may be detected with a hybridizationprobe having a nucleotide sequence outside the region used for theinhibitory double-stranded RNA, or translated polypeptide may bedetected with an antibody raised against the polypeptide sequence ofthat region.

The RNA may be introduced in an amount which allows delivery of at leastone copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000copies per cell) of material may yield more effective inhibition; lowerdoses may also be useful for specific applications.

In an exemplary aspect, the efficacy of an RNAi agent of the invention(e.g., an siRNA targeting an htt target sequence) is tested for itsability to specifically degrade mutant mRNA (e.g., htt mRNA and/or theproduction of huntingtin protein) in cells, in particular, in neurons(e.g., striatal or cortical neuronal clonal lines and/or primaryneurons). Also suitable for cell-based validation assays are otherreadily transfectable cells, for example, HeLa cells or COS cells. Cellsare transfected with human wild type or mutant cDNAs (e.g., human wildtype or mutant huntingtin cDNA). Standard siRNA, modified siRNA orvectors able to produce siRNA from U-looped mRNA are co-transfected.Selective reduction in target mRNA (e.g., huntingtin mRNA) and/or targetprotein (e.g., huntingtin protein) is measured. Reduction of target mRNAor protein can be compared to levels of target mRNA or protein in theabsence of an RNAi agent or in the presence of an RNAi agent that doesnot target htt mRNA. Exogenously-introduced mRNA or protein (orendogenous mRNA or protein) can be assayed for comparison purposes. Whenutilizing neuronal cells, which are known to be somewhat resistant tostandard transfection techniques, it may be desirable to introduce RNAiagents (e.g., siRNAs) by passive uptake.

Recombinant Adeno-Associated Viruses and Vectors

In certain exemplary embodiments, recombinant adeno-associated viruses(rAAVs) and their associated vectors can be used to deliver one or moresiRNAs into cells, e.g., neural cells (e.g., brain cells). AAV is ableto infect many different cell types, although the infection efficiencyvaries based upon serotype, which is determined by the sequence of thecapsid protein. Several native AAV serotypes have been identified, withserotypes 1-9 being the most commonly used for recombinant AAV. AAV-2 isthe most well-studied and published serotype. The AAV-DJ system includesserotypes AAV-DJ and AAV-DJ/8. These serotypes were created through DNAshuffling of multiple AAV serotypes to produce AAV with hybrid capsidsthat have improved transduction efficiencies in vitro (AAV-DJ) and invivo (AAV-DJ/8) in a variety of cells and tissues.

In particular embodiments, widespread central nervous system (CNS)delivery can be achieved by intravascular delivery of recombinantadeno-associated virus 7 (rAAV7), RAAV9 and rAAV10, or other suitablerAAVs (Zhang et al. (2011) Mol. Ther. 19(8):1440-8. doi:10.1038/mt.2011.98. Epub 2011 May 24). rAAVs and their associatedvectors are well-known in the art and are described in US PatentApplications 2014/0296486, 2010/0186103, 2008/0269149, 2006/0078542 and2005/0220766, each of which is incorporated herein by reference in itsentirety for all purposes.

rAAVs may be delivered to a subject in compositions according to anyappropriate methods known in the art. An rAAV can be suspended in aphysiologically compatible carrier (i.e., in a composition), and may beadministered to a subject, i.e., a host animal, such as a human, mouse,rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig,hamster, chicken, turkey, a non-human primate (e.g., Macaque) or thelike. In certain embodiments, a host animal is a non-human host animal.

Delivery of one or more rAAVs to a mammalian subject may be performed,for example, by intramuscular injection or by administration into thebloodstream of the mammalian subject. Administration into thebloodstream may be by injection into a vein, an artery, or any othervascular conduit. In certain embodiments, one or more rAAVs areadministered into the bloodstream by way of isolated limb perfusion, atechnique well known in the surgical arts, the method essentiallyenabling the artisan to isolate a limb from the systemic circulationprior to administration of the rAAV virions. A variant of the isolatedlimb perfusion technique, described in U.S. Pat. No. 6,177,403, can alsobe employed by the skilled artisan to administer virions into thevasculature of an isolated limb to potentially enhance transduction intomuscle cells or tissue. Moreover, in certain instances, it may bedesirable to deliver virions to the central nervous system (CNS) of asubject. By “CNS” is meant all cells and tissue of the brain and spinalcord of a vertebrate. Thus, the term includes, but is not limited to,neuronal cells, glial cells, astrocytes, cerebrospinal fluid (CSF),interstitial spaces, bone, cartilage and the like. Recombinant AAVs maybe delivered directly to the CNS or brain by injection into, e.g., theventricular region, as well as to the striatum (e.g., the caudatenucleus or putamen of the striatum), spinal cord and neuromuscularjunction, or cerebellar lobule, with a needle, catheter or relateddevice, using neurosurgical techniques known in the art, such as bystereotactic injection (see, e.g., Stein et al., J Virol 73:3424-3429,1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidson et al., Nat.Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther.11:2315-2329, 2000).

The compositions of the invention may comprise an rAAV alone, or incombination with one or more other viruses (e.g., a second rAAV encodinghaving one or more different transgenes). In certain embodiments, acomposition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more differentrAAVs each having one or more different transgenes.

An effective amount of an rAAV is an amount sufficient to target infectan animal, target a desired tissue. In some embodiments, an effectiveamount of an rAAV is an amount sufficient to produce a stable somatictransgenic animal model. The effective amount will depend primarily onfactors such as the species, age, weight, health of the subject, and thetissue to be targeted, and may thus vary among animal and tissue. Forexample, an effective amount of one or more rAAVs is generally in therange of from about 1 ml to about 100 ml of solution containing fromabout 10⁹ to 10¹⁶ genome copies. In some cases, a dosage between about10¹¹ to 10¹² rAAV genome copies is appropriate. In certain embodiments,10¹² rAAV genome copies is effective to target heart, liver, andpancreas tissues. In some cases, stable transgenic animals are producedby multiple doses of an rAAV.

In some embodiments, rAAV compositions are formulated to reduceaggregation of AAV particles in the composition, particularly where highrAAV concentrations are present (e.g., about 10¹³ genome copies/mL ormore). Methods for reducing aggregation of rAAVs are well known in theart and, include, for example, addition of surfactants, pH adjustment,salt concentration adjustment, etc. (See, e.g., Wright et al. (2005)Molecular Therapy 12:171-178, the contents of which are incorporatedherein by reference.)

“Recombinant AAV (rAAV) vectors” comprise, at a minimum, a transgene andits regulatory sequences, and 5′ and 3′ AAV inverted terminal repeats(ITRs). It is this recombinant AAV vector which is packaged into acapsid protein and delivered to a selected target cell. In someembodiments, the transgene is a nucleic acid sequence, heterologous tothe vector sequences, which encodes a polypeptide, protein, functionalRNA molecule (e.g., siRNA) or other gene product, of interest. Thenucleic acid coding sequence is operatively linked to regulatorycomponents in a manner which permits transgene transcription,translation, and/or expression in a cell of a target tissue.

The AAV sequences of the vector typically comprise the cis-acting 5′ and3′ inverted terminal repeat (ITR) sequences (See, e.g., B. J. Carter, in“Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168(1990)). The ITR sequences are usually about 145 basepairs in length. Incertain embodiments, substantially the entire sequences encoding theITRs are used in the molecule, although some degree of minormodification of these sequences is permissible. The ability to modifythese ITR sequences is within the skill of the art. (See, e.g., textssuch as Sambrook et al, “Molecular Cloning. A Laboratory Manual”, 2ded., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher etal., J Virol., 70:520 532 (1996)). An example of such a moleculeemployed in the present invention is a “cis-acting” plasmid containingthe transgene, in which the selected transgene sequence and associatedregulatory elements are flanked by the 5′ and 3′ AAV ITR sequences. TheAAV ITR sequences may be obtained from any known AAV, includingmammalian AAV types described further herein.

IX. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a diseaseor disorder caused, in whole or in part, by a gain of function mutantprotein. In one embodiment, the disease or disorder is a trinucleotiderepeat disease or disorder. In another embodiment, the disease ordisorder is a polyglutamine disorder. In a preferred embodiment, thedisease or disorder is a disorder associated with the expression ofhuntingtin and in which alteration of huntingtin, especially theamplification of CAG repeat copy number, leads to a defect in huntingtingene (structure or function) or huntingtin protein (structure orfunction or expression), such that clinical manifestations include thoseseen in Huntington's disease patients.

“Treatment,” or “treating,” as used herein, is defined as theapplication or administration of a therapeutic agent (e.g., a RNA agentor vector or transgene encoding same) to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has the disease or disorder, a symptom of disease ordisorder or a predisposition toward a disease or disorder, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease or disorder, the symptoms of the diseaseor disorder, or the predisposition toward disease.

In one aspect, the invention provides a method for preventing in asubject, a disease or disorder as described above, by administering tothe subject a therapeutic agent (e.g., an RNAi agent or vector ortransgene encoding same). Subjects at risk for the disease can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe disease or disorder, such that the disease or disorder is preventedor, alternatively, delayed in its progression.

Another aspect of the invention pertains to methods treating subjectstherapeutically, i.e., alter onset of symptoms of the disease ordisorder. In an exemplary embodiment, the modulatory method of theinvention involves contacting a cell expressing a gain-of-functionmutant with a therapeutic agent (e.g., a RNAi agent or vector ortransgene encoding same) that is specific for a target sequence withinthe gene (e.g., SEQ ID NOs:1, 2 or 3), such that sequence specificinterference with the gene is achieved. These methods can be performedin vitro (e.g., by culturing the cell with the agent) or, alternatively,in vivo (e.g., by administering the agent to a subject).

With regards to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics,” as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype,” or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the target genemolecules of the present invention or target gene modulators accordingto that individual's drug response genotype. Pharmacogenomics allows aclinician or physician to target prophylactic or therapeutic treatmentsto patients who will most benefit from the treatment and to avoidtreatment of patients who will experience toxic drug-related sideeffects.

Therapeutic agents can be tested in an appropriate animal model. Forexample, an RNAi agent (or expression vector or transgene encoding same)as described herein can be used in an animal model to determine theefficacy, toxicity, or side effects of treatment with said agent.Alternatively, a therapeutic agent can be used in an animal model todetermine the mechanism of action of such an agent. For example, anagent can be used in an animal model to determine the efficacy,toxicity, or side effects of treatment with such an agent.Alternatively, an agent can be used in an animal model to determine themechanism of action of such an agent.

A pharmaceutical composition containing an RNA silencing agent of theinvention can be administered to any patient diagnosed as having or atrisk for developing a neurological disorder, such as Huntington'sdisease. In one embodiment, the patient is diagnosed as having aneurological disorder, and the patient is otherwise in general goodhealth. For example, the patient is not terminally ill, and the patientis likely to live at least 2, 3, 5 or more years following diagnosis.The patient can be treated immediately following diagnosis, or treatmentcan be delayed until the patient is experiencing more debilitatingsymptoms, such as motor fluctuations and dyskinesis in Parkinson'sdisease patients. In another embodiment, the patient has not reached anadvanced stage of the disease.

An RNA silencing agent modified for enhance uptake into neural cells canbe administered at a unit dose less than about 1.4 mg per kg ofbodyweight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005,0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight, andless than 200 nmole of RNA agent (e.g., about 4.4×10¹⁶ copies) per kg ofbodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75,0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of RNAsilencing agent per kg of bodyweight. The unit dose, for example, can beadministered by injection (e.g., intravenous or intramuscular,intrathecally, or directly into the brain), an inhaled dose, or atopical application. Particularly preferred dosages are less than 2, 1,or 0.1 mg/kg of body weight.

Delivery of an RNA silencing agent directly to an organ (e.g., directlyto the brain) can be at a dosage on the order of about 0.00001 mg toabout 3 mg per organ, or preferably about 0.0001-0.001 mg per organ,about 0.03-3.0 mg per organ, about 0.1-3.0 mg per eye or about 0.3-3.0mg per organ. The dosage can be an amount effective to treat or preventa neurological disease or disorder, e.g., Huntington's disease. In oneembodiment, the unit dose is administered less frequently than once aday, e.g., less than every 2, 4, 8 or 30 days. In another embodiment,the unit dose is not administered with a frequency (e.g., not a regularfrequency). For example, the unit dose may be administered a singletime. In one embodiment, the effective dose is administered with othertraditional therapeutic modalities.

In one embodiment, a subject is administered an initial dose, and one ormore maintenance doses of an RNA silencing agent. The maintenance doseor doses are generally lower than the initial dose, e.g., one-half lessof the initial dose. A maintenance regimen can include treating thesubject with a dose or doses ranging from 0.01 μg to 1.4 mg/kg of bodyweight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg ofbodyweight per day. The maintenance doses are preferably administered nomore than once every 5, 10, or 30 days. Further, the treatment regimenmay last for a period of time which will vary depending upon the natureof the particular disease, its severity and the overall condition of thepatient. In preferred embodiments the dosage may be delivered no morethan once per day, e.g., no more than once per 24, 36, 48, or morehours, e.g., no more than once every 5 or 8 days. Following treatment,the patient can be monitored for changes in his condition and foralleviation of the symptoms of the disease state. The dosage of thecompound may either be increased in the event the patient does notrespond significantly to current dosage levels, or the dose may bedecreased if an alleviation of the symptoms of the disease state isobserved, if the disease state has been ablated, or if undesiredside-effects are observed.

The effective dose can be administered in a single dose or in two ormore doses, as desired or considered appropriate under the specificcircumstances. If desired to facilitate repeated or frequent infusions,implantation of a delivery device, e.g., a pump, semi-permanent stent(e.g., intravenous, intraperitoneal, intracisternal or intracapsular),or reservoir may be advisable. In one embodiment, a pharmaceuticalcomposition includes a plurality of RNA silencing agent species. Inanother embodiment, the RNA silencing agent species has sequences thatare non-overlapping and non-adjacent to another species with respect toa naturally occurring target sequence. In another embodiment, theplurality of RNA silencing agent species is specific for differentnaturally occurring target genes. In another embodiment, the RNAsilencing agent is allele specific. In another embodiment, the pluralityof RNA silencing agent species target two or more target sequences(e.g., two, three, four, five, six, or more target sequences).

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the compound of the invention is administered inmaintenance doses, ranging from 0.01 μg to 100 g per kg of body weight(see U.S. Pat. No. 6,107,094).

The concentration of the RNA silencing agent composition is an amountsufficient to be effective in treating or preventing a disorder or toregulate a physiological condition in humans. The concentration oramount of RNA silencing agent administered will depend on the parametersdetermined for the agent and the method of administration, e.g. nasal,buccal, or pulmonary. For example, nasal formulations tend to requiremuch lower concentrations of some ingredients in order to avoidirritation or burning of the nasal passages. It is sometimes desirableto dilute an oral formulation up to 10-100 times in order to provide asuitable nasal formulation.

Certain factors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of an RNA silencing agent caninclude a single treatment or, preferably, can include a series oftreatments. It will also be appreciated that the effective dosage of anRNA silencing agent for treatment may increase or decrease over thecourse of a particular treatment. Changes in dosage may result andbecome apparent from the results of diagnostic assays as describedherein. For example, the subject can be monitored after administering anRNA silencing agent composition. Based on information from themonitoring, an additional amount of the RNA silencing agent compositioncan be administered.

Dosing is dependent on severity and responsiveness of the diseasecondition to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of disease state is achieved. Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient. Persons of ordinary skill can easily determine optimum dosages,dosing methodologies and repetition rates. Optimum dosages may varydepending on the relative potency of individual compounds, and cangenerally be estimated based on EC50s found to be effective in in vitroand in vivo animal models. In some embodiments, the animal modelsinclude transgenic animals that express a human gene, e.g., a gene thatproduces a target RNA, e.g., an RNA expressed in a neural cell. Thetransgenic animal can be deficient for the corresponding endogenous RNA.In another embodiment, the composition for testing includes an RNAsilencing agent that is complementary, at least in an internal region,to a sequence that is conserved between the target RNA in the animalmodel and the target RNA in a human.

X. Pharmaceutical Compositions and Methods of Administration

The invention pertains to uses of the above-described agents forprophylactic and/or therapeutic treatments as described Infra.Accordingly, the modulators (e.g., RNAi agents) of the present inventioncan be incorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the nucleic acidmolecule, protein, antibody, or modulatory compound and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, intraperitoneal, intramuscular, oral (e.g., inhalation),transdermal (topical), and transmucosal administration. In certainexemplary embodiments, a pharmaceutical composition of the invention isdelivered to the cerebrospinal fluid (CSF) by a route of administrationthat includes, but is not limited to, intrastriatal (IS) administration,intracerebroventricular (ICV) administration and intrathecal (IT)administration (e.g., via a pump, an infusion or the like). Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfate; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous, IS, ICV and/or ITadministration, suitable carriers include physiological saline,bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). In all cases, the composition must besterile and should be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

The RNA silencing agents can also be administered by transfection orinfection using methods known in the art, including but not limited tothe methods described in McCaffrey et al. (2002), Nature, 418(6893),38-9 (hydrodynamic transfection); Xia et al. (2002), Nature Biotechnol.,20(10), 1006-10 (viral-mediated delivery); or Putnam (1996), Am. J.Health Syst. Pharm. 53(2), 151-160, erratum at Am. J. Health Syst.Pharm. 53(3), 325 (1996).

The RNA silencing agents can also be administered by any method suitablefor administration of nucleic acid agents, such as a DNA vaccine. Thesemethods include gene guns, bio injectors, and skin patches as well asneedle-free methods such as the micro-particle DNA vaccine technologydisclosed in U.S. Pat. No. 6,194,389, and the mammalian transdermalneedle-free vaccination with powder-form vaccine as disclosed in U.S.Pat. No. 6,168,587. Additionally, intranasal delivery is possible, asdescribed in, inter alia, Hamajima et al. (1998), Clin. Immunol.Immunopathol., 88(2), 205-10. Liposomes (e.g., as described in U.S. Pat.No. 6,472,375) and microencapsulation can also be used. Biodegradabletargetable microparticle delivery systems can also be used (e.g., asdescribed in U.S. Pat. No. 6,471,996).

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit large therapeutic indices are preferred. Althoughcompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the EC50 (i.e., the concentration ofthe test compound which achieves a half-maximal response) as determinedin cell culture. Such information can be used to more accuratelydetermine useful doses in humans. Levels in plasma may be measured, forexample, by high performance liquid chromatography.

The pharmaceutical compositions can be included in a container, pack ordispenser together with optional instructions for administration.

As defined herein, a therapeutically effective amount of a RNA silencingagent (i.e., an effective dosage) depends on the RNA silencing agentselected. For instance, if a plasmid encoding shRNA is selected, singledose amounts in the range of approximately 1 μg to 1000 mg may beadministered; in some embodiments, 10, 30, 100 or 1000 μg may beadministered. In some embodiments, 1-5 g of the compositions can beadministered. The compositions can be administered one from one or moretimes per day to one or more times per week; including once every otherday. The skilled artisan will appreciate that certain factors mayinfluence the dosage and timing required to effectively treat a subject,including but not limited to the severity of the disease or disorder,previous treatments, the general health and/or age of the subject, andother diseases present. Moreover, treatment of a subject with atherapeutically effective amount of a protein, polypeptide, or antibodycan include a single treatment or, preferably, can include a series oftreatments.

The nucleic acid molecules of the invention can be inserted intoexpression constructs, e.g., viral vectors, retroviral vectors,expression cassettes, or plasmid viral vectors, e.g., using methodsknown in the art, including but not limited to those described in Xia etal., (2002), Supra. Expression constructs can be delivered to a subjectby, for example, inhalation, orally, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994), Proc. Natl. Acad. Sci. USA, 91,3054-3057). The pharmaceutical preparation of the delivery vector caninclude the vector in an acceptable diluent, or can comprise a slowrelease matrix in which the delivery vehicle is imbedded. Alternatively,where the complete delivery vector can be produced intact fromrecombinant cells, e.g., retroviral vectors, the pharmaceuticalpreparation can include one or more cells which produce the genedelivery system.

The nucleic acid molecules of the invention can also include smallhairpin RNAs (shRNAs), and expression constructs engineered to expressshRNAs. Transcription of shRNAs is initiated at a polymerase III (polIII) promoter, and is thought to be terminated at position 2 of a4-5-thymine transcription termination site. Upon expression, shRNAs arethought to fold into a stem-loop structure with 3′ UU-overhangs;subsequently, the ends of these shRNAs are processed, converting theshRNAs into siRNA-like molecules of about 21 nucleotides. Brummelkamp etal. (2002), Science, 296, 550-553; Lee et al, (2002). supra; Miyagishiand Taira (2002), Nature Biotechnol., 20, 497-500; Paddison et al.(2002), supra; Paul (2002), supra; Sui (2002) supra; Yu et al. (2002),supra.

The expression constructs may be any construct suitable for use in theappropriate expression system and include, but are not limited toretroviral vectors, linear expression cassettes, plasmids and viral orvirally-derived vectors, as known in the art. Such expression constructsmay include one or more inducible promoters, RNA Pol III promotersystems such as U6 snRNA promoters or H1 RNA polymerase III promoters,or other promoters known in the art. The constructs can include one orboth strands of the siRNA. Expression constructs expressing both strandscan also include loop structures linking both strands, or each strandcan be separately transcribed from separate promoters within the sameconstruct. Each strand can also be transcribed from a separateexpression construct, Tuschl (2002), Supra.

In certain exemplary embodiments, a composition that includes an RNAsilencing agent of the invention can be delivered to the nervous systemof a subject by a variety of routes. Exemplary routes includeintrathecal, parenchymal (e.g., in the brain), nasal, and oculardelivery. The composition can also be delivered systemically, e.g., byintravenous, subcutaneous or intramuscular injection, which isparticularly useful for delivery of the RNA silencing agents toperipheral neurons. A preferred route of delivery is directly to thebrain, e.g., into the ventricles or the hypothalamus of the brain, orinto the lateral or dorsal areas of the brain. The RNA silencing agentsfor neural cell delivery can be incorporated into pharmaceuticalcompositions suitable for administration.

For example, compositions can include one or more species of an RNAsilencing agent and a pharmaceutically acceptable carrier. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic, intranasal, transdermal), oral orparenteral. Parenteral administration includes intravenous drip,subcutaneous, intraperitoneal or intramuscular injection, intrathecal,or intraventricular (e.g., intracerebroventricular) administration. Incertain exemplary embodiments, an RNA silencing agent of the inventionis delivered across the Blood-Brain-Barrier (BBB) suing a variety ofsuitable compositions and methods described herein.

The route of delivery can be dependent on the disorder of the patient.For example, a subject diagnosed with Huntington's disease can beadministered an anti-htt RNA silencing agent of the invention directlyinto the brain (e.g., into the globus pallidus or the corpus striatum ofthe basal ganglia, and near the medium spiny neurons of the corposstriatum). In addition to an RNA silencing agent of the invention, apatient can be administered a second therapy, e.g., a palliative therapyand/or disease-specific therapy. The secondary therapy can be, forexample, symptomatic (e.g., for alleviating symptoms), neuroprotective(e.g., for slowing or halting disease progression), or restorative(e.g., for reversing the disease process). For the treatment ofHuntington's disease, for example, symptomatic therapies can include thedrugs haloperidol, carbamazepine, or valproate. Other therapies caninclude psychotherapy, physiotherapy, speech therapy, communicative andmemory aids, social support services, and dietary advice.

An RNA silencing agent can be delivered to neural cells of the brain.Delivery methods that do not require passage of the composition acrossthe blood-brain barrier can be utilized. For example, a pharmaceuticalcomposition containing an RNA silencing agent can be delivered to thepatient by injection directly into the area containing thedisease-affected cells. For example, the pharmaceutical composition canbe delivered by injection directly into the brain. The injection can beby stereotactic injection into a particular region of the brain (e.g.,the substantia nigra, cortex, hippocampus, striatum, or globuspallidus). The RNA silencing agent can be delivered into multipleregions of the central nervous system (e.g., into multiple regions ofthe brain, and/or into the spinal cord). The RNA silencing agent can bedelivered into diffuse regions of the brain (e.g., diffuse delivery tothe cortex of the brain).

In one embodiment, the RNA silencing agent can be delivered by way of acannula or other delivery device having one end implanted in a tissue,e.g., the brain, e.g., the substantia nigra, cortex, hippocampus,striatum or globus pallidus of the brain. The cannula can be connectedto a reservoir of RNA silencing agent. The flow or delivery can bemediated by a pump, e.g., an osmotic pump or minipump, such as an Alzetpump (Durect, Cupertino, Calif.). In one embodiment, a pump andreservoir are implanted in an area distant from the tissue, e.g., in theabdomen, and delivery is effected by a conduit leading from the pump orreservoir to the site of release. Devices for delivery to the brain aredescribed, for example, in U.S. Pat. Nos. 6,093,180, and 5,814,014.

An RNA silencing agent of the invention can be further modified suchthat it is capable of traversing the blood brain barrier. For example,the RNA silencing agent can be conjugated to a molecule that enables theagent to traverse the barrier. Such modified RNA silencing agents can beadministered by any desired method, such as by intraventricular orintramuscular injection, or by pulmonary delivery, for example.

In certain embodiments, exosomes are used to deliver an RNA silencingagent of the invention. Exosomes can cross the BBB and deliver siRNAs,antisense oligonucleotides, chemotherapeutic agents and proteinsspecifically to neurons after systemic injection (See, Alvarez-Erviti L,Seow Y, Yin H, Betts C, Lakhal S, Wood M J. (2011). Delivery of siRNA tothe mouse brain by systemic injection of targeted exosomes. NatBiotechnol. 2011 April; 29(4):341-5. doi: 10.1038/nbt.1807;El-Andaloussi S, Lee Y, Lakhal-Littleton S, Li J, Seow Y, Gardiner C,Alvarez-Erviti L, Sargent I L, Wood M J. (2011). Exosome-mediateddelivery of siRNA in vitro and in vivo. Nat Protoc. 2012 December;7(12):2112-26. doi: 10.1038/nprot.2012.131; EL Andaloussi S, Mäger I,Breakefield X O, Wood M J. (2013). Extracellular vesicles: biology andemerging therapeutic opportunities. Nat Rev Drug Discov. 2013 May;12(5):347-57. doi: 10.1038/nrd3978; El Andaloussi S, Lakhal S, Mager I,Wood M J. (2013). Exosomes for targeted siRNA delivery across biologicalbarriers. Adv Drug Deliv Rev. 2013 March; 65(3):391-7. doi:10.1016/j.addr.2012.08.008).

In certain embodiments, one or more lipophilic molecules are used toallow delivery of an RNA silencing agent of the invention past the BBB(Alvarez-Ervit (2011)). The RNA silencing agent would then be activated,e.g., by enzyme degradation of the lipophilic disguise to release thedrug into its active form.

In certain embodiments, one or more receptor-mediated permeablizingcompounds can be used to increase the permeability of the BBB to allowdelivery of an RNA silencing agent of the invention. These drugsincrease the permeability of the BBB temporarily by increasing theosmotic pressure in the blood which loosens the tight junctions betweenthe endothelial cells ((El-Andaloussi (2012)). By loosening the tightjunctions normal intravenous injection of an RNA silencing agent can beperformed.

In certain embodiments, nanoparticle-based delivery systems are used todeliver an RNA silencing agent of the invention across the BBB. As usedherein, “nanoparticles” refer to polymeric nanoparticles that aretypically solid, biodegradable, colloidal systems that have been widelyinvestigated as drug or gene carriers (S. P. Egusquiaguirre, M. Igartua,R. M. Hernandez, and J. L. Pedraz, “Nanoparticle delivery systems forcancer therapy: advances in clinical and preclinical research,” Clinicaland Translational Oncology, vol. 14, no. 2, pp. 83-93, 2012). Polymericnanoparticles are classified into two major categories, natural polymersand synthetic polymers. Natural polymers for siRNA delivery include, butare not limited to, cyclodextrin, chitosan, and atelocollagen (Y. Wang,Z. Li, Y. Han, L. H. Liang, and A. Ji, “Nanoparticle-based deliverysystem for application of siRNA in vivo,” Current Drug Metabolism, vol.11, no. 2, pp. 182-196, 2010). Synthetic polymers include, but are notlimited to, polyethyleneimine (PEI), poly(dl-lactide-co-glycolide)(PLGA), and dendrimers, which have been intensively investigated (X.Yuan, S. Naguib, and Z. Wu, “Recent advances of siRNA delivery bynanoparticles,” Expert Opinion on Drug Delivery, vol. 8, no. 4, pp.521-536, 2011). For a review of nanoparticles and other suitabledelivery systems, See Jong-Min Lee, Tae-Jong Yoon, and Young-Seok Cho,“Recent Developments in Nanoparticle-Based siRNA Delivery for CancerTherapy,” BioMed Research International, vol. 2013, Article ID 782041,10 pages, 2013. doi:10.1155/2013/782041 (incorporated by reference inits entirety.)

An RNA silencing agent of the invention can be administered ocularly,such as to treat retinal disorder, e.g., a retinopathy. For example, thepharmaceutical compositions can be applied to the surface of the eye ornearby tissue, e.g., the inside of the eyelid. They can be appliedtopically, e.g., by spraying, in drops, as an eyewash, or an ointment.Ointments or droppable liquids may be delivered by ocular deliverysystems known in the art such as applicators or eye droppers. Suchcompositions can include mucomimetics such as hyaluronic acid,chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinylalcohol), preservatives such as sorbic acid, EDTA or benzylchroniumchloride, and the usual quantities of diluents and/or carriers. Thepharmaceutical composition can also be administered to the interior ofthe eye, and can be introduced by a needle or other delivery devicewhich can introduce it to a selected area or structure. The compositioncontaining the RNA silencing agent can also be applied via an ocularpatch.

In general, an RNA silencing agent of the invention can be administeredby any suitable method. As used herein, topical delivery can refer tothe direct application of an RNA silencing agent to any surface of thebody, including the eye, a mucous membrane, surfaces of a body cavity,or to any internal surface. Formulations for topical administration mayinclude transdermal patches, ointments, lotions, creams, gels, drops,sprays, and liquids. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable. Topical administration can also be used as a means toselectively deliver the RNA silencing agent to the epidermis or dermisof a subject, or to specific strata thereof, or to an underlying tissue.

Compositions for intrathecal or intraventricular (e.g.,intracerebroventricular) administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Compositions for intrathecal or intraventricularadministration preferably do not include a transfection reagent or anadditional lipophilic moiety besides, for example, the lipophilic moietyattached to the RNA silencing agent.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir. Forintravenous use, the total concentration of solutes should be controlledto render the preparation isotonic.

An RNA silencing agent of the invention can be administered to a subjectby pulmonary delivery. Pulmonary delivery compositions can be deliveredby inhalation of a dispersion so that the composition within thedispersion can reach the lung where it can be readily absorbed throughthe alveolar region directly into blood circulation. Pulmonary deliverycan be effective both for systemic delivery and for localized deliveryto treat diseases of the lungs. In one embodiment, an RNA silencingagent administered by pulmonary delivery has been modified such that itis capable of traversing the blood brain barrier.

Pulmonary delivery can be achieved by different approaches, includingthe use of nebulized, aerosolized, micellular and dry powder-basedformulations. Delivery can be achieved with liquid nebulizers,aerosol-based inhalers, and dry powder dispersion devices. Metered-dosedevices are preferred. One of the benefits of using an atomizer orinhaler is that the potential for contamination is minimized because thedevices are self-contained. Dry powder dispersion devices, for example,deliver drugs that may be readily formulated as dry powders. An RNAsilencing agent composition may be stably stored as lyophilized orspray-dried powders by itself or in combination with suitable powdercarriers. The delivery of a composition for inhalation can be mediatedby a dosing timing element which can include a timer, a dose counter,time measuring device, or a time indicator which when incorporated intothe device enables dose tracking, compliance monitoring, and/or dosetriggering to a patient during administration of the aerosol medicament.

The types of pharmaceutical excipients that are useful as carriersinclude stabilizers such as human serum albumin (HSA), bulking agentssuch as carbohydrates, amino acids and polypeptides; pH adjusters orbuffers; salts such as sodium chloride; and the like. These carriers maybe in a crystalline or amorphous form or may be a mixture of the two.

Bulking agents that are particularly valuable include compatiblecarbohydrates, polypeptides, amino acids or combinations thereof.Suitable carbohydrates include monosaccharides such as galactose,D-mannose, sorbose, and the like; disaccharides, such as lactose,trehalose, and the like; cyclodextrins, such as2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such asraffinose, maltodextrins, dextrans, and the like; alditols, such asmannitol, xylitol, and the like. A preferred group of carbohydratesincludes lactose, trehalose, raffinose maltodextrins, and mannitol.Suitable polypeptides include aspartame. Amino acids include alanine andglycine, with glycine being preferred.

Suitable pH adjusters or buffers include organic salts prepared fromorganic acids and bases, such as sodium citrate, sodium ascorbate, andthe like; sodium citrate is preferred.

An RNA silencing agent of the invention can be administered by oral andnasal delivery. For example, drugs administered through these membraneshave a rapid onset of action, provide therapeutic plasma levels, avoidfirst pass effect of hepatic metabolism, and avoid exposure of the drugto the hostile gastrointestinal (GI) environment. Additional advantagesinclude easy access to the membrane sites so that the drug can beapplied, localized and removed easily. In one embodiment, an RNAsilencing agent administered by oral or nasal delivery has been modifiedto be capable of traversing the blood-brain barrier.

In one embodiment, unit doses or measured doses of a composition thatinclude RNA silencing agents are dispensed by an implanted device. Thedevice can include a sensor that monitors a parameter within a subject.For example, the device can include a pump, such as an osmotic pump and,optionally, associated electronics.

An RNA silencing agent can be packaged in a viral natural capsid or in achemically or enzymatically produced artificial capsid or structurederived therefrom.

XI. Kits

In certain other aspects, the invention provides kits that include asuitable container containing a pharmaceutical formulation of an RNAsilencing agent, e.g., a double-stranded RNA silencing agent, or sRNAagent, (e.g., a precursor, e.g., a larger RNA silencing agent which canbe processed into a sRNA agent, or a DNA which encodes an RNA silencingagent, e.g., a double-stranded RNA silencing agent, or sRNA agent, orprecursor thereof). In certain embodiments the individual components ofthe pharmaceutical formulation may be provided in one container.Alternatively, it may be desirable to provide the components of thepharmaceutical formulation separately in two or more containers, e.g.,one container for an RNA silencing agent preparation, and at leastanother for a carrier compound. The kit may be packaged in a number ofdifferent configurations such as one or more containers in a single box.The different components can be combined, e.g., according toinstructions provided with the kit. The components can be combinedaccording to a method described herein, e.g., to prepare and administera pharmaceutical composition. The kit can also include a deliverydevice.

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods described hereinmay be made using suitable equivalents without departing from the scopeof the embodiments disclosed herein. Having now described certainembodiments in detail, the same will be more clearly understood byreference to the following example, which is included for purposes ofillustration only and is not intended to be limiting.

EXAMPLES Example 1 Reduction of Huntingtin in Both Primary Neurons andMouse Brain with Unformulated, Stabilized, Hydrophobic siRNAs

The use of hydrophobically modified ASO-siRNA hybrids, which have thepotential to offer both better efficacy and distribution in vivo andknockdown in primary neurons in vitro, was explored. The huntingtin genewas used as a target for mRNA knockdown. Huntington's disease ismonogenic (Mangiarini, L. et al. Exon 1 of the HTT gene with an expandedCAG repeat is sufficient to cause a progressive neurological phenotypein transgenic mice. Cell 87, 493-506 (1996)) with a number of cellularmechanisms leading to disease pathology (Zuccato, C., Valenza, M. &Cattaneo, E. Molecular Mechanisms and Potential Therapeutical Targets inHuntington's Disease. Physiological Reviews 90, 905-981 (2010)) makingit an excellent candidate for possible future oligonucleotidetherapeutics.

A panel of hydrophobically modified siRNAs targeting the Huntingtin genewas developed. Efficacy and potency in was observed both in primaryneurons in vitro, and in vivo in mouse brain upon a single low doseinjection without any formulation for delivery. These compounds combinea number of different chemical and structural modifications found bothin earlier model siRNAs and hsiRNAs, as well as in ASOs. Theseproperties, which include stabilizing base modifications, cholesterolconjugation, and a fully phosphorothioated single stranded tail, makethese hsiRNAs excellent tools for studying gene function inhard-to-target primary cells and organs that can be adapted for use in anumber of different biologically relevant systems.

1.1 hsiRNA—Hydrophobically Modified siRNA/Antisense Hybrids wereEfficiently Internalized by Primary Neurons

The hsiRNAs were asymmetric compounds, with a short duplex region (15base-pairs) and single-stranded fully phosphorothioated tail. Allpyrimidines in these compounds were 2′-Fluoro and 2′-O-Methyl modified(providing stabilization), and the 3′ end of the passenger strand wasconjugated to TEG-Cholesterol (FIG. 1A, FIG. 8) 13. The cholesterolconjugate enabled quick membrane association, while the single strandedphosphorothioated tail was necessary for cellular internalization by amechanism similar to the one used by conventional antisenseoligonucleotides. Addition of Cy3-labeled hsiRNA to primary corticalneurons resulted in immediate (within minutes) cellular association(FIG. 1B). Interestingly, the uptake was first observed preferentiallyin dendrites, followed by re-localization to the cellular body (FIG. 9).The uptake was uniform across all cells in the dish, affirming efficientinternalization.

1.2 Identification of hsiRNAs Targeting Huntingtin

A panel of 94 hsiRNA compounds (FIG. 8) targeting huntingtin mRNA wasdesigned and synthesized. These sequences spanned the gene and wereselected to comply with standard siRNA design parameters (Birmingham, A.et al. A protocol for designing siRNAs with high functionality andspecificity. Nat Protoc 2, 2068-2078 (2007)) including assessment of GCcontent, specificity and low seed compliment frequency (Anderson, E. M.et al. Experimental validation of the importance of seed complementfrequency to siRNA specificity. RNA 14, 853-861 (2008)), elimination ofsequences containing miRNA seeds, and examination of thermodynamic bias(Khvorova, A., Reynolds, A. & Jayasena, S. D. Functional siRNAs andmiRNAs Exhibit Strand Bias. Cell 115, 209-216 (2003); Schwarz, D. S. etal. Asymmetry in the Assembly of the RNAi Enzyme Complex. Cell 115,199-208 (2003)). More than 50% of bases were chemically modified, toprovide in vivo stability and minimization of immune response (Judge,A., Bola, G., Lee, A. & MacLachlan, I. Design of NoninflammatorySynthetic siRNA Mediating Potent Gene Silencing in Vivo. MolecularTherapy 13, 494-505 (2006)). The modifications imposed additionalrestrictions on sequence space, reducing the hit rate. Impact onHuntingtin mRNA expression was measured after 72 hours exposure to 1.5μM hsiRNA (passive uptake, no formulation) in HeLa cells by QUANTIGENEassay (FIG. 2), with 7% of sequences showing more than 70% silencing.Functional target sites were spread across the gene with the exceptionof the distal part of the 3′UTR, later explained by preferentialexpression of the shorter htt isoform in HeLa cells (Li, S. H. et al.Huntington's disease gene (IT15) is widely expressed in human and rattissues. NEURON 11, 985-993 (1993)). IC50 values were identified forsixteen active sequences, selected based on primary screen activity andcross-species conservation (FIG. 10). IC50 values ranged from 90 to 766nM in passive uptake (no formulation) and from 4 to 91 pM inlipid-mediated uptake (FIG. 8). Fully chemically-optimized activecompounds were readily identified, indicating that a much smallerlibrary should be sufficient in future screens for other genes, althoughhit rate is likely to be variable from target to target. The hsiRNAtargeting position 10150 (HTT10150 (i.e., 5′ CAGUAAAGAGAUUAA 3′ (SEQ IDNO:1))) was used for further studies. To ensure that the hsiRNA chemicalscaffold did not negatively impact efficacy and potency of HTT10150, themodified and unmodified versions of the compound were tested in bothpassive and lipid-mediated silencing assays (FIG. 3). As expected, onlythe modified sequence was successful at cellular delivery and Httsilencing by passive uptake (IC50=82.2 nM), while both the modified andunmodified compounds showed similar IC50 values in lipid mediateddelivery (4 pM and 13 pM respectively) suggesting that the hsiRNAscaffold modifications did not interfere with RNA-Induced SilencingComplex (RISC) loading.

1.3 Potent and Specific Gene Silencing with Unformulated hsiRNAs inPrimary Neurons

HTT10150 was further tested for mRNA silencing in primary neuronsisolated from FVBN mice. Efficacy was seen at both 72 hours and one weekfollowing simple unformulated compound addition to cortical neurons(FIG. 4A) with maximum silencing (70%) observed at the 1.25 μMconcentration. HTT10150 also showed similar silencing in primarystriatal neurons (FIG. 4B). Protein levels were measured after one weekby Western blot (FIG. 14), confirming mRNA data with 85% reduction ofprotein upon treatment with 1.25 μM of compound (FIG. 4C). Thehousekeeping genes (PPIB, GAPDH) and overall cell viability, measured byALAMARBLUE Assay (FIGS. 11B and 14), were not affected at theseconcentrations. In other experiments, a slight impact on cell viabilitywas observed at 3 μM.

To evaluate duration of effect upon a single HTT10150 treatment, thesilencing was measured at one week, two week, and three week intervals(FIG. 4D). The half-life of the loaded RISC complex was weeks (Song, E.et al. Sustained Small Interfering RNA-Mediated Human ImmunodeficiencyVirus Type 1 Inhibition in Primary Macrophages. Journal of Virology 77,7174-7181 (2003)), and silencing was expected to be long lasting innon-dividing cells. Indeed, single treatment with hsiRNAs was sufficientto induce htt silencing at all times tested. Three weeks was the longestthe primary neurons could be maintained in culture. Other systems willbe used for longer-term experiments.

To demonstrate the general applicability of hsiRNAs as a tool forneuronal gene silencing, and to confirm this chemistry scaffold as validfor neuronal delivery, similar experiments were performed with severalother hsiRNAs targeting HTT and with one targeting the house-keepinggene PPIB (Cyclophilin B) (FIGS. 11A and 13). Silencing as high as 70and 90% was achieved with HTT and PPIB, respectively.

In summary, these data demonstrate that hydrophobically modified siRNAis a simple and straightforward approach for gene silencing in primaryneurons, and can be adapted for multiple gene targets.

1.4 hsiRNA Distribution In Vivo in Mouse Brain Upon Single Injection

hsiRNAs are efficiently internalized by different types of neurons invitro. The selected hsiRNA, HTT10150, was further evaluated for itspotential to silence gene expression in the brain in vivo. To determinethe distribution profile of HTT10150 upon in vivo administration, 12.5μg of Cy3 labelled hsiRNA (See FIG. 8 for sequence) was injectedintrastriatally and, after 24 hours, the brain was perfused, sectioned,and oligonucleotide distribution was visualized by fluorescencemicroscopy (Leica DM5500—DFC365FX). The artificial CSF injected samplesprocessed concurrently were used to set up microscopic imaging settingsto control for background tissue epifluorescence.

The majority of compound showed a steep gradient of diffusion away fromthe injection site, with most of the ipsilateral striatum being covered(FIG. 5A, 5B). Interestingly, hsiRNAs were detected on the non-injectedside (contralateral) side of the brain (both cortex and striatum),although relative concentrations appeared much lower. Highermagnification images showed significant association of hsiRNA with fibertracks, most likely due to the presence of a hydrophobic modification.This aspect of hsiRNA may make it useful as a labelling reagent tovisualize brain signalling architecture (FIG. 5C, 5D). In addition tofiber tracks and neurite labelling, hsiRNA could be detected as punctatestaining in the perinuclear space of different cell types, includingneurons, as evident from co-localization with NeuN (neuronal marker)stained cells (FIG. 5E) only 24 hours after injection.

The effect of vitamin D on hsiRNA distribution is depicted in FIGS. 79and 80.

1.5 hsiRNA Efficacy In Vivo in Mouse Brain Upon Single Injection

To determine HTT10150 efficacy in vivo, wild type FVBN mice were dosedintrastriatally with a single injection of between 3 and 25 μg (0.1-0.9mg/kg) of compound and mRNA silencing was examined both ipsilateral andcontralateral to the injection site. Eight animals were dosed pertreatment group and three individual punches were taken from each sideof the striatum for mRNA and protein quantification. Level of huntingtinexpression were measured by QUANTIGENE Assay and normalized to ahousekeeping gene (details in Online methods).

Statistical analysis was performed by one-way ANOVA comparison againstCSF or PBS control with Bonferroni corrections for repeat measures usingGraphPad Prism (Online methods for details). All groups inducedsilencing that was significant against CSF, PBS, and non-targetingcontrol treated animals. Raw Data from the 24 individual punches pertreatment group (8 animals, 3 punches per animal) can be seen in FIG.15. At the site of administration (ipsilateral side), dose-dependentsilencing reaching statistical significance was observed at allconcentrations. The 25 μg treatment induced 77% silencing (p<0.0001),and the 12.5 μg treatment was repeated with two groups of animals ondifferent days and showed statistically significant silencing of 66% and42% (FIG. 6).

While initial distribution studies showed a steep gradient of diffusionaway from the injection site with a minimal amount of compound migratingto the contralateral side, treatment with the higher doses of 25 μg and12.5 μg resulted in statistically significant silencing (p<0.0001) onthe non-injected side. However, the level of silencing was significantlyless (only 36% for the 25 μg group) than on the treated side of thebrain.

In summary, these data show that a single intrastriatal injection ofhsiRNA is sufficient to induce potent gene silencing around the site ofadministration. This effect was reproducible across different treatmentgroups and independent experiments.

1.6 Neuronal Viability Following Single hsiRNA Injection in Mouse Brain

Cholesterol modification of non-modified, naked siRNA has previouslybeen used for improvement of siRNA brain distribution, with toxicity athigh doses being identified as a potential limitation. To evaluate thedegree of non-specific chemistry related effects on the brain, DARPP32expression, an established marker for dopamine receptor expression onmedium spiny neurons in the striatum and representative of neuronalviability, was investigated. Additionally, potential induction of animmune response was performed by assessing the extent of microgliaactivation upon hsiRNA injection.

No significant impact on DARPP32 expression was observed for doses up to12.5 μg suggesting persistent neuronal viability (FIGS. 7A, 7B, 16).Similarly, minimal microglial activation was visualized at the 12.5 μgdose (FIG. 7C, 7D) indicative of a limited immune response in thepresence of the modified hsiRNA. The 25 μg dose did induce somereduction in DARPP32 just around the site of injection indicative oftoxicity and establishing the maximum dose levels for this chemicalscaffold upon the indicated route of administration. A 10-12.5 μg singleadministration of hsiRNA efficiently silenced HTT mRNA in three, wellpowered, independent studies with robust silencing of 62, 42 and 52%without toxicity. These data indicate that this technology can be widelyused for functional studies of other neurologically significant targets.

1.7 Further Characterization in Neurons

Sustained silencing was achieved for 21 days interminally-differentiated neurons (FIG. 24). A silencing plateau wasobserved with RNAi (cytoplasmic) but not RNaseH (predominantly nuclear)compounds (FIG. 25). The observed plateau was specific to the htt gene.Approximately 60% of htt mRNA localized in the nuclei (FIG. 26).

Probe sets were validated in neurons (FIG. 27). A majority of thedetected signal was specific to htt mRNA. A high fraction of yellow(co-localized staining) areas were observed. Without intending to bebound by scientific theory, the high degree of red signal may be relatedto uneven concentrations of the two probed sets.

Additional probe sets were validated for intron 60-61 in neurons (FIG.28). Intron-specific probes showed one to two yellow dots in the nucleispecific to transcription sites. Exon-specific probes showed a higherdegree of overlap.

Htt mRNA nuclear localization was specific to neurons and not tofibroblasts (FIG. 29). HsiRNA^(HTT) treatment of cortical neuronspreferentially eliminated cytoplasmic htt mRNA (FIGS. 30 and 31).

Close to complete HTT protein silencing was observed in primary corticalneurons (FIG. 32).

Direct injection of HTT10150 caused no detectable changes in neuronalnumbers (FIG. 33). Cholesterol-hsiRNA exhibited a small area of toxicityadjacent to the injection site (FIG. 34).

FIGS. 58-60 disclose hsiRNA efficacy in wild-type and Q140 primaryhippocampal neurons.

1.8 Discussion

This study demonstrates that the use of hydrophobically modified siRNAfor delivery to primary cells is a valuable tool to enable functionaland genomic studies of neuronal pathways and neurological disorders.

The ability to cause gene silencing in primary neurons without the useof toxic formulation has a significant impact on neuroscience research,facilitating a more in depth study of neurological disorders in thecontext of primary cell lines, and ultimately providing a more relevantunderstanding of in vivo function and pathology. Most neuronal studiesare done in stable cell lines due to ease of delivery and cellmaintenance, but using artificial cell systems can lead to artifacts inthe data that can be attributed to manipulation of these cell lines, aproblem that can be avoided by using primary cells (Cheung, Y.-T. et al.Effects of all-trans-retinoic acid on human SH-SY5Y neuroblastoma as invitro model in neurotoxicity research. NeuroToxicology 30, 127-135(2009); Gilany, K. et al. The proteome of the human neuroblastoma cellline SH-SY5Y: An enlarged proteome. Biochimica et Biophysica Acta(BBA)—Proteins and Proteomics 1784, 983-985 (2008); Lopes, F. M. et al.Comparison between proliferative and neuron-like SH-SY5Y cells as an invitro model for Parkinson disease studies. Brain Research 1337, 85-94(2010); Zhang, W. et al. Cyclohexane 1,3-diones and their inhibition ofmutant SOD1-dependent protein aggregation and toxicity in PC12 cells.BIOORGANIC & MEDICINAL CHEMISTRY 1-17 (2011).doi:10.1016/j.bmc.2011.11.039). Current methods for delivering siRNA toprimary neurons include the use of lentiviral vectors, Adeno-AssociatedViruses (AAV), or Lipofectamine™-mediated transfection (Karra, D. &Dahm, R. Transfection Techniques for Neuronal Cells. Journal ofNeuroscience 30, 6171-6177 (2010)). By conjugating a hydrophobic moietysuch as cholesterol directly to the siRNA itself and by utilizing anadditional single stranded phosphorothioated tail for enhanced uptake,it has been demonstrated herein that, not only can siRNA be deliveredefficiently into primary neurons in vitro with minimal toxicity, butalso remains a potent silencer of mRNA.

Without intending to be bound by scientific theory, one of the majoradvantages of RNAi over antisense technology is that the loaded RISC isexpected to remain active for a long period of time in non-dividingcells (Bartlett, D. W. Insights into the kinetics of siRNA-mediated genesilencing from live-cell and live-animal bioluminescent imaging. NucleicAcids Research 34, 322-333 (2006)). Additionally, a limited number ofloaded RISCs are sufficient for the induction of RNAi-mediated silencing(Stalder, L. et al. The rough endoplasmatic reticulum is a centralnucleation site of siRNA-mediated RNA silencing. The EMBO Journal 32,1115-1127 (2013)). The data presented herein demonstrates silencing forup to three weeks in vitro in primary cortical neurons upon a singletreatment with hsiRNA, supporting the notion that RNAi-mediatedsilencing can be both efficient and long lasting. The data presentedherein also shows that these compounds can be used to target multipleregions in two different genes, which demonstrates the adaptability ofhsiRNA for the study of alternative neurological pathways and diseases.

While a single intra-striatal injection of hsiRNA resulted in potentgene silencing near the injection site in vivo, the effect was notevenly spread throughout the brain. Although limited, spread to otherareas of the brain (demonstrated by in vivo efficacy studies) could behappening through a number of mechanisms. These include movement in theCSF, spread via fiber tracts which were shown to have a large visualdensity of Cy3-labeled hsiRNA in distribution studies, or possiblythrough retrograde transport (Stewart, G. R. & Sah, D. RetrogradeTransport of siRNA and Therapeutic Uses to Treat Neurological Disorders.United States Patent Application Publication US 2008/0039415 A1, 1-18(2008)), although further studies will be conducted to determine theactual mechanism.

The technology presented herein is useful for understanding functionalgenomics of particular brain regions, as well as for studyingrelationships between brain regions. Additionally, the study of someneurological disorders (for example memory disorders (Samuelson, K. W.Post-traumatic stress disorder and declarative memory functioning: areview. Dialogues in Clinical Neuroscience 13, 346-351 (2011))) canbenefit from limited and regionally targeted distribution and silencing.However, due to its distribution profile, hsiRNA as it currently existsis not a viable therapeutic for general neurological disorders likeHuntington's disease. Multiple injections may work to increase overallsilencing in small rodents, but in order to adapt this technology foruse in larger animal brains and humans, and to achieve even andwidespread distribution, other chemical modifications and therapeuticmethods of delivery will be utilized. There are a number of ways inwhich this might be approached. First, chemical adjustments to thehsiRNA composition itself can be made. These include conjugating it to adifferent lipid, supplementing the backbone with additionalphosphorothioate groups, or by addition of hydrophobic moieties to thenucleotides themselves (Vaught, J. D., Dewey, T. & Eaton, B. E. T7 RNAPolymerase Transcription with 5-Position Modified UTP Derivatives. J.Am. Chem. Soc. 126, 11231-11237 (2004)). All of these modificationscould support a range of hydrophobicities that would allow for moreimproved distribution across a larger distance. Increasedbioavailability could also be achieved with different modes of injectionsuch as into the CSF instead of intrastriatally, increasing thelikelihood of exposure to the whole brain. However, delivery via the CSFcould favor localization of hsiRNA to brain regions other than thestriatum, making it a less than ideal delivery method for the treatmentof Huntington's disease. Another possibility is formulated delivery bypackaging these hydrophobically modified siRNAs into exosomes andliposomes (less toxic than current Lipofectamine™ formulations) andusing these natural and synthetic nanocarriers to deliver cargo in amore evenly distributed fashion (Alvarez-Erviti, L. et al. Delivery ofsiRNA to the mouse brain by systemic injection of targeted exosomes. NatBiotechnol 1-7 (2011). doi:10.1038/nbt.1807; Marcus, M. & Leonard, J.FedExosomes: Engineering Therapeutic Biological Nanoparticles that TrulyDeliver. Pharmaceuticals 6, 659-680 (2013)). However, potency andefficacy of the delivered hsiRNA still needs to be validated for thesemethods.

In conclusion, HTT10150 was efficient for targeting huntingtin mRNA inprimary neurons in vitro and locally in the mouse brain in vivo. Thiscompound did not require any formulation for delivery to primary cellsand enabled gene functional studies for huntingtin as well as othertargets, making it a very useful tool for the study of neurologicaldisorders. Potential advances to this technology should allow for hsiRNAto function as a therapeutic treatment for Huntington's disease as wellas other neurological diseases in the future.

1.9 Methods

Cell Culture

HeLa cells were maintained in DMEM (Corning Cellgro) supplemented with10% fetal bovine serum (Gibco) and 100 U/mL penicillin/streptomycin(Invitrogen) and grown at 37° C. and 5% CO₂. Cells were split every 2-5days up to passage 15 and then discarded.

Cell Culture for Passive Uptake

Cells were plated in DMEM with 6% FBS at 10,000 cells/well in 96-welltissue culture treated plates. hsiRNA was diluted in OptiMEM (Gibco) to2× final concentration and 50 μL diluted hsiRNA was added to 50 μL ofcells for 3% FBS final. Cells were incubated for 72 hours at 37° C. and5% CO₂.

Cell Culture for Lipid-Mediated Uptake

Cells were plated in DMEM with 6% FBS at 10,000 cells/well in 96-welltissue culture treated plates. hsiRNA was diluted in OptiMEM to 4× finalconcentration. LIPOFECTAMINE RNAIMAX Transfection Reagent (Invitrogen#13778150) was diluted to 4× final concentration (final=0.3 μL/25μL/well). RNAIMAX and hsiRNA were mixed 1:1 and 50 μL was added to 50 μLof cells for 3% FBS final. Cells were incubated for 72 hours at 37° C.and 5% CO₂.

Preparation of Primary Neurons

Primary cortical neurons were obtained from E15.5 mouse embryos of WT(FVBN) mice. Pregnant females were anesthetized by IP injection ofAvertin (250 mg/kg weight) followed by cervical dislocation. Embryoswere removed and transferred into a Petri dish with ice-cold DMEM/F12medium (Invitrogen). Brains were removed and meninges were carefullydetached. Cortices were isolated and transferred into a 1.5-ml tube withpre-warmed papain solution for 25 minutes at 37° C. and 5% CO₂ todissolve tissue. Papain solution was prepared as follows: papain(Worthington #54N15251) was dissolved in 2 mL HibernateE (Brainbits) and1 mL EBSS (Worthington). Separately, DNase (Worthington #54M15168) wasre-suspended in 0.5 mL HibernateE. Then, 0.25 mL of re-suspended DNasewas transferred to re-suspended papain for the final solution. After the25 minute incubation, papain solution was removed and 1 mL NbActiv4(Brainbits) supplemented with 2.5% FBS was added to the tissue. Thecortices were then dissociated by pipetting up and down with a firepolished, glass Pasteur pipet. Cortical neurons were counted and platedat 1×10⁶ cells/ml. For live-cell imaging studies, culture plates werepre-coated with poly-L-lysine (Sigma #P4707) and 2×10⁵ cells were addedto the glass center of each dish. For silencing assays, neurons wereplated on poly-L-lysine pre-coated 96-well plates (BD BIOCOAT #356515)at 1×10⁵ cells per well. After overnight incubation at 37° C. and 5% CO₂an equal volume of NbActiv4 (Brainbits) supplemented with anti-mitotics,0.484 μL/mL of 5′UtP (Sigma #U6625) and 0.2402 μL/mL of 5′FdU (Sigma#F3503), to prevent the growth of non-neuronal cells, was added toneuronal cultures. Half of the volume of media was replaced every 48hours (with new NbActiv4 with anti-mitotics) until the neurons weretreated with siRNA. Once the cells were treated, media was not removed,only added. All subsequent media additions contained anti-mitotics.

mRNA Quantification

mRNA was quantified using the QuantiGene 2.0 Assay (Affymetrix #QS0011).Cells were lysed in 250 μL diluted lysis mixture (Affymetrix #13228), 1part lysis mixture, 2 parts H₂O, with 0.167 μg/μL proteinase K(Affymetrix #QS0103) for 30 minutes at 55° C. Cell lysates were mixedthoroughly and 40 μL (approximately 8000 cells) of lysate were added tothe capture plate along with 40 μL additional diluted lysis mixturewithout proteinase K. Probe sets were diluted as specified in theAffymetrix protocol. For HeLa cells, 20 μL of human HTT or PPIB probeset (Affymetrix #SA-50339, #SA-10003) was added to appropriate wells fora final volume of 100 μL. For primary neurons, 20 μL of mouse HTT orPPIB probe set (Affymetrix #SB-14150, #SB-10002) was used.

Tissues were treated similarly, using 300 μL of Homogenizing Buffer(Affymetrix #10642) with 2 μg/μL proteinase K for a 5 mg tissue punch.Tissues were then homogenized in 96-well plate format on the QIAGENTissueLyser II and 40 μL were added to the capture plate. Probe setswere diluted as specified in the Affymetrix protocol and 60 μL of eitherHTT or PPIB probe sets (Affymetrix #SB-14150, #SB-10002) were added toeach well of the capture plate for a final volume of 100 μL. For DARPP32quantification, only 10 μL of tissue sample and 30 μL of homogenizingbuffer were added to each well with 60 μL of mouse Ppp1r1b probe set(Affymetrix #SB-21622). Signal was amplified according to the Affymetrixprotocol. Luminescence was detected on either the Veritas Luminometer orthe Tecan M 1000.

Live Cell Staining

To monitor live cell hsiRNA uptake, cells were plated at a density of2×10⁵ cells per 35 mm glass-bottom dish as described in the preparationof primary neurons above. Prior to imaging, cell nuclei were stained inphenol red free NbActiv4 using NUCBLUE (Molecular Probes by LifeTechnologies #R37605) as indicated by the manufacturer. Imaging wasperformed in phenol red free NbActiv4. Cells were treated with 0.5 μM ofCy3-labeled hsiRNA, and live cell imaging was performed over time. Alllive cell confocal images were acquired with a Zeiss confocal microscopeand images were processed using ImageJ (1.47v) software.

Immunohistochemistry/Immunofluorescence

For distribution studies, brains were injected with 1 nmol (12.5 μg) ofCy3-labeled hsiRNA. After 24 hours, mice were sacrificed and brains wereremoved and sent to the DERC Morphology Core at UMASS Medical School tobe embedded in paraffin and sliced into 4 μm sections and mounted onglass slides. Sections were de-parafinized for 8 minutes in xylene twotimes. Sections were then rehydrated with serial ethanol dilutions(100%, 95%, 80%) for 4 minutes each, then washed twice for two minuteswith PBS. For NueN staining, slides were boiled for 5 minutes in antigenretrieval buffer and then left to sit at room temperature for 20minutes, followed by a 5-minute wash with PBS. Slides were then blockedwith 5% normal goat serum in PBS+0.05% Tween20 for 1 hour and washedonce with PBS+0.05% Tween20 for 5 minutes. Primary antibody (1:1000dilution in PBS+0.05% Tween20) was added to slides for a 1 hourincubation followed by three 5-minute washes with PBS+0.05% Tween20.Secondary antibody (1:1000 dilution in PBS+0.05% Tween20) was added toslides for a 30-minute incubation in the dark followed by three 5-minutewashes with PBS+0.05% Tween20. Slides were then stained with DAPI(Molecular Probes by Life Technologies #D3571), diluted to 250 ng/mL inPBS, for one minute followed by three 1-minute washes with PBS. Mountingmedia and coverslips were applied to slides and left to dry over nightbefore imaging on Leica DM5500—DFC365FX microscope at indicatedmagnification.

For toxicity and microglia activation studies extracted, perfused brainswere sliced into 40 μm sections on the Leica 2000T Vibratome in ice coldPBS. Immunohistochemistry was performed on every 6th section againstDARPP32 (Millipore, 1:10,000 dilution) and IBA-1 (Millipore, 1:500dilution). Sections were mounted and visualized by light microscopy.Four images were taken at 20× in the striatum of both injected andnon-injected sides of each section. The number of DARPP32 positiveneurons was quantified using ImageJ. Activated microglia was quantifiedby morphology of stained cells for IBA-1.

Animals, Stereotaxic Injections

Wild-type (FVBN) mice received microinjections by stereotactic placementinto the right striata (coordinates (relative to bregma) were 1.0 mmanterior, 2.0 mm lateral, and 3.0 mm ventral). Animals were deeplyanesthetized prior to injection with 1.2% Avertin. For both toxicity(DARPP32) and efficacy studies, mice received injections of either PBSor artificial cerebrospinal fluid (2 μL per striata, N=8 mice), 12.5 μgof NTC hsiRNA (2 μL of 500 μM stock solution per striata, N=8 mice), 25μg of HTT10150 hsiRNA (2 μL of 1 mM stock solution per striata, N=8mice), 12.5 μg of HTT10150 hsiRNA (2 μL of 500 μM stock solution perstriata, N=16 mice total, two sets of 8 mice on two different days), 6.3μg of HTT10150 hsiRNA (2 μL of 250 μM stock solution per striata, N=8mice), or 3.1 μg of HTT10150 hsiRNA (2 μL of 125 μM stock solution perstriata, N=8 mice) and euthanized 5 days later. Brains were harvestedand three 300 μm coronal sections were made. One 2 mm punch was takenper side (injected and non-injected) for each section and placed inRNAlater (Ambion #AM7020) for 24 hours at 4° C. Each punch was processedas an individual sample for the QuantiGene assay analysis. All animalprocedures were approved by the University of Massachusetts MedicalSchool Institutional Animal Care and Use Committee (IACUC, protocolnumber A-2411).

Statistical Analysis

Data analyses were done using GraphPad Prism 6 version 6.04 software(GraphPad Software, Inc., San Diego, Calif.). For concentrationdependent curve IC50s, a curve was fitted using log(inhibitor) vs.response—variable slope (four parameters). The bottom of the curve wasset to be no less than zero and the top of the curve was set to be nogreater than 100. For each independent mouse experiment, the level ofknockdown at each dose was normalized to the mean of the control group,which was the non-injected side of the PBS or artificial CSF groups, sothat all data were expressed as a proportion of the control. In vivodata were analyzed using the Kruskal-Wallis test (one-way ANOVA) withBonferroni corrections for multiple comparisons. Differences in allcomparisons were considered significant at P-values less than 0.05.

Cell Culture for Passive Uptake (Primary Screen and Dose Response)

Cells were plated in DMEM (Gibco) with 6% FBS (Gibco) at 10,000cells/well in 96-well tissue culture treated plates. HsiRNA was dilutedin OptiMEM (Gibco) to 2× final concentration and 50 uL diluted hsiRNAwas added to 50 μL of cells for 3% FBS final. Cells were incubated for72 hours at 37 C and 5% CO₂.

Cell Culture for Lipid-Mediated Uptake

Cells were plated in DMEM (Gibco) with 6% FBS (Gibco) at 10,000cells/well in 96-well tissue culture treated plates. HsiRNA was dilutedin OptiMEM (Gibco) to 4× final concentration. LIPOFECTAMINE RNAIMAXTransfection Reagent (Invitrogen CAT #13778150) was diluted to 4× finalconcentration (final=0.3 μL/25 μL/well). RNAIMAX and hsiRNA were mixed1:1 and 50 μL was added to 50 uL of cells for 3% FBS final. Cells wereincubated for 72 hours at 37 C and 5% CO₂.

mRNA Quantification

mRNA was quantified using the QuantiGene 2.0 Assay (Affymetrix QS0011).Cells were lysed in 250 μL diluted lysis mixture, 1 part lysis mixture,2 parts H2O, with 0.167 μg/μL proteinase K (Affymetrix QS0103) for 30minutes at 55 C. Cell lysates were mixed thoroughly and 40 μL (˜8000cells) of lysate were added to capture plate along with 40 μL additionaldiluted lysis mixture without proteinase K. Tissues were treatedsimilarly, using 300 μL of Homoginizing Buffer (Affymetrix) with 2 μg/μLproteinase K for a 5 mg tissue punch. Tissues were then homogenized in96-well plate format on Qaigen TissueLyzer and 40 μL were added tocapture plate. Probe sets were diluted as specified in Affymetrixprotocol and 20 μL of either HTT or PPIB probes (Affymetrix: SA-50339,SA-10003) were added to each well of capture plate for final volume of100 μL. Signal was amplified according to manufacture protocol.Luminescence was detected on either the Veritas Luminometer or the TecanM 1000.

Live Cell Staining and Brain Sections Immunostaining

For live cell uptake monitoring, cells were plated at a density of 2×10⁵cells per 35 mm glass-bottom dish and grown overnight. Prior to imaging,cell organelles were stained in HBSS (Gibco) using staining reagentspurchased from Life Technologies unless specified: cell nuclei,endoplasmic reticulum and lysosomes were respectively stained using theNUCBLUE Live READYPROBE, ER-TRACKER Green (Bodipy FL Glibenclamide) andLYSOTRACKER Deep Red reagents as indicated by the manufacturer. Imagingwas performed in non-supplemented DMEM without phenol red (Invitrogen).Cells were treated with 0.5 μM of Cy3-labeled hsiRNA, and live cellimaging was performed over time.

Confocal Imaging

All confocal images were acquired with a CSU10B Spinning Disk ConfocalSystem scan head (Solamere Technology Group) mounted on a TE-200E2inverted microscope (Nikon) with a 60× Plan/APO oil lens and a CoolsnapHQ2 camera (Roper). Images were processed using ImageJ (1.47v) software.Number of neurons without or with hsiRNA was counted using ImageJsoftware. Brain sections images were acquired with a z-axis spacing of 1μm.

Example 2 hsiRNA Retention and Distribution is Directly Related toHydrophobicity

Although FM-hsiRNAs showed improved retention and accumulation in brainand spinal cord and induce maximal silencing at 10-fold lower doses thanpartially stabilized hsiRNAs, they were largely retained near theinjection site (FIG. 102; Chol-hsiRNA). It was hypothesized that thelimited distribution of hsiRNAs could result from preferential bindingof hsiRNA to lipid-enriched myelin and myelinated structures due to thestrong hydrophobicity of the cholesterol conjugate, and that tuning thehydrophobicity of the hsiRNA conjugates would improve distributionthrough the spinal cord and brain. To test the idea, a panel ofnaturally occurring, hydrophobic molecules capable of active neuronaltrafficking, was screened including: (i) neuroactive steroids, i.e.,endogenous steroids that traverse the blood-brain barrier and bind avariety of gated-ion channels and neuronal-expressed receptors(Rupprecht R. Neuroactive steroids: mechanisms of action andneuropsychopharmacological properties. Psychoneuroendocrinology. 2003;28:139-68. PMID: 12510009), including GABA (Lan N C, Gee K W.Neuroactive steroid actions at the GABAA receptor. Hormones andbehavior. 1994; 28:537-44. PMID: 7729823); (ii)gangliosides—neuroprotective glycolipids critical for neuronalplasticity and repair (Aureli M, Mauri L, Ciampa M G, Prinetti A,Toffano G, Secchieri C, Sonnino S. GM1 Ganglioside: Past Studies andFuture Potential. Molecular neurobiology. 2015. PMID: 25762012); and(iii) endocannabinoid-like long-chain polyunsaturated fattyacids—neuromodulatory lipids recognized by receptors involved inappetite, pain, mood, and memory (Dyall S C. Long-chain omega-3 fattyacids and the brain: a review of the independent and shared effects ofEPA, DPA and DHA. Frontiers in aging neuroscience. 2015; 7:52. PMID:25954194; PMCID: PMC4404917; Janssen C I, Kiliaan A J. Long-chainpolyunsaturated fatty acids (LCPUFA) from genesis to senescence: theinfluence of LCPUFA on neural development, aging, and neurodegeneration.Progress in lipid research. 2014; 53:1-17. PMID: 24334113; Figueroa J D,De Leon M. Neurorestorative targets of dietary long-chain omega-3 fattyacids in neurological injury. Molecular neurobiology. 2014; 50:197-213.PMID: 24740740; PMCID: PMC4183712).

The most robust approach for synthesis of oligonucleotide conjugates wasto attach the activated conjugate to an amino-modified support. Thestructure and length of the linker were varied (e.g., branched) and thesupport was functionalized whenever feasible. Variations of thesynthetic approaches outlined in FIGS. 93 and 94 were used to synthesizehsiRNAs conjugated to cortisol, docosahexaenoic acid (DHA), calciferol,cholesterol, and GM1 ganglioside (FIGS. 98 and 99). All compounds wereHPLC-purified and their identities were confirmed by mass spectrometry.The calciferol-functionalized support was unstable, resulting in amixture of several variants that were tested in vivo (described infra).

As expected, the compounds showed different degrees of hydrophobicitybased on retention time during reverse phase chromatography. Injectionof Cy3-labeled hsiRNA conjugates into striata or ICV (FIG. 92) ofwild-type mice revealed varying degrees of hsiRNA distribution andretention that strongly correlated with hydrophobicity. Non-conjugatedor linker-only hsiRNAs showed minimal retention in the brain (similar tothat of antisense oligonucleotides) and the most hydrophobic compounds,cholesterol and GM1, were primarily retained near the site of injection.Optimal retention/distribution was achieved with DHA and calciferolconjugates (infra), which have intermediate hydrophobicity profiles.DHA-hsiRNA was studied in detail and showed great efficacy andunprecedented safety (therapeutic index>20-fold) (Nikan et al., 2016;Molecular Therapy, in revision, see Appendix). In summary, the datapresented herein show that tuning the hydrophobicity of conjugates is avalid strategy to identify conjugates that support optimal retention,distribution, and safety in brain tissues.

GM1-hsiRNA was efficiently internalized and induced huntingtin mRNAsilencing in primary cortical neurons (FIG. 108). GM1-hsiRNA displayedlimited distribution in mouse brain upon intrastriatal injection (FIG.109).

Example 3 DHA-hsiRNA

Partially-modified hsiRNAs demonstrated a short duration of effect andno systemic exposure (FIGS. 35A-C). Metabolic stabilization was furtherexplored (FIG. 36). Full metabolic stabilization did not interfere withRISC entry of hsiRNAs (FIG. 37). Fully metabolically stabilized hsiRNA(FM-hsiRNA) enhanced local delivery and distribution and enabled alonger duration of effect (FIGS. 38, 39A-B, 91, 110 and 111 The term“nucleoside”).

Naturally-occurring lipids (i.e., glycosphingolipids, polyunsaturatedfatty acids, secosteroids, steroid hormones and sterol lipids) wereinvestigated as hsiRNA bioconjugates (FIG. 40). Lipid bioconjugates hada pronounced effect on hsiRNA^(HTT) sense strand hydrophobicity.

A study was designed to explore in vivo distribution of hsiRNAconjugates. Intrastriatal unilateral injection (2 nmol/2 μl) of FVBN WTmice with P2-stabilized siRNA CY3 conjugates in aCSF was performed. 48hours post-injection, animals were perfused with PBS and 10% formalin.Their brains were removed and post-fixed for 48 hours. 4 μm coronal andsagittal slices were prepared and stained with DAPI. Imaging wasperformed on a Leica DM 5500 fluorescent microscope (CY3 and DAPI). Itwas determined that hsiRNA hydrophobicity was directly correlated withbrain distribution and retention (FIG. 41). A key property was a balancebetween distribution and retention.

Docosahexaenoic acid (DHA)—hsiRNAs were synthesized (FIG. 42). DHA is anomega-3 fatty acid that is a primary component of the human brain (70%).DHA crosses the bold brain barrier (BBB) and is actively internalized byneurons and other cell types. It is a non-toxic supplement clinicallyshown to improve cognitive function in HD and ALS patients. DHA issignificantly less hydrophobic that cholesterol.

DHA-hsiRNA and chol-hsiRNA were shown to be internalized into primarycortical neurons (FIG. 43). DHA-hsiRNA co-localized with neurons andastrocytes (FIG. 44) and was localized to the perinuclear region ofstriatal neurons (chol-hsiRNA was undetectable in striatal neurons)(FIG. 45). DHA-hsiRNA co-localized with neurons and astrocytes in thecortex following a single intrastriatal injection (FIG. 46). DHA-hsiRNAlocalized to the perinuclear region in cortical neurons, whilechol-hsiRNA was undetectable (FIG. 47). DHA-hsiRNA efficientlydistributed throughout the brain and silenced genes in both the striatumand the cortex (FIG. 57).

DHA-hsiRNA showed robust efficacy in the striatum and the cortex (FIGS.48 and 49). Up to 200 μg DHA-hsiRNA had no effect on DARPP-32 levels,indicating compound safety (FIG. 50). In contrast, 25 μg (1 mg/kg) wasthe maximum tolerated intrastriatal dose of chol-hsiRNA. Up to 200 μgDHA-hsiRNA caused no significant increase in activated microglia,indicating minimal immune stimulation (FIG. 51).

hsiRNA allows for simple and efficient gene silencing in primary neuronsin vivo in the brain. Oligonucleotide hydrophobicity defines braintissue retention and distribution. Oligonucleotide chemistry was shownto impact cellular delivery and distribution (FIGS. 52-56). DHA-hsiRNAconjugates represent a new class of oligonucleotides with wide in vivoefficacy and a wide therapeutic index.

Example 4 PC-DHA-hsiRNA (PC-DHA-hsiRNA)

Encouraged by the wide therapeutic index of DHA-hsiRNAs, DHA and relatedconjugates were investigated in more detail. Circulating DHA is mostlypresent as a lysophosphatidylcholine ester, which is the only formactively trafficked through the blood-brain barrier via the specifictransporter Mfsd2a (Nguyen L N, Ma D, Shui G, Wong P, Cazenave-GassiotA, Zhang X, Wenk M R, Goh E L, Silver D L. Mfsd2a is a transporter forthe essential omega-3 fatty acid docosahexaenoic acid. Nature. 2014;509:503-6. PMID: 24828044).

The lysophosphatidylcholine ester of DHA is unstable, so alysophosphatidylcholine (PC) amide of DHA was synthesized (FIGS. 93, 94,100 and 101). PC-DHA is a metabolically stable analog compatible withsolid-phase oligonucleotide synthesis. Its identity was confirmed by NMRand mass spectrometry. Testing the idea that lysophosphatidylcholineshould improve trafficking of DHA-hsiRNA, it was determined that thatPC-DHA-hsiRNAs showed a wider distribution and increased efficacy inbrain tissue than do DHA-hsiRNAs (FIGS. 92 and 103). Importantly, as aclass, DHA conjugates showed a wide therapeutic index with no obviousinnate immune activation or neuronal degeneration at concentrations20-fold higher than the minimum effective dose (FIG. 92). Comparatively,Chol-hsiRNA showed significant toxicity at 25 μg injections (FIG. 92C).Lastly, a bolus CSF (ICV) infusion supported wide distribution in thebrain, covering striatum, cortex and even reaching more posterior andventral regions of the brain (FIG. 92). Due to its exceptionalcharacteristics, PC-DHA-hsiRNA was selected as a lead chemistry toinvestigate.

PC-DHA is a metabolically active analogue of DHA (FIG. 62).PC-DHA-hsiRNA demonstrates enhanced neuronal silencing in vitro,enhanced brain distribution and enhanced in vivo potency (with no signsof toxicity) relative to DHA-hsiRNA.

PC-DHA-hsiRNA and chol-hsiRNA were each shown to efficiently silenceboth mutant and wild-type htt mRNA (FIG. 61). Chol-hsiRNA demonstratedtoxicity (3 out of 6 animals died). The living animals demonstrated verylow (3-fold over background) human htt expression.

PC-DHA-hsiRNA, when delivered to primary neurons, demonstrated enhancedpotency relative to DHA-hsiRNA (FIG. 63). Although chol-hsiRNA was moreeffective in decreasing htt gene expression in primary neurons (FIG.64), PC-DHA-hsiRNA showed superior brain retention and widerdistribution (FIG. 65).

PC-DHA-hsiRNA showed approximately 80% silencing in mouse striatum aftera single interstitial (IS) injection (FIG. 66) and showed approximately60% silencing in mouse cortex after a single IS injection (FIG. 67).There was no indication of toxicity. Silencing was limited to injectedside of the brain.

The kidney is the main target of PC-DHA-hsiRNA (FIG. 107). PC-DHA-hsiRNAaccumulated in the proximal convoluted tubules.

Example 5 Discovery of Di-hsiRNAs

Branched oligonucleotides represent a novel class of oligonucleotidetherapeutics. Two to eight oligonucleotides were attached togetherthrough a hydrophobic linker, with 2-3 oligonucleotides attachedtogether being preferred. Substantial chemical stabilization wastypically used (at least 40% bases modified, fully modified preferred.Single stranded phosphorothioated tail of 2-20 was typically used (with8-10 preferred).

The discovery of di-branched hsiRNA (di-hsiRNA) compounds was pureserendipity. Calciferol readily oxidizes and the solid support proved tobe unstable, complicating QC and purification. A pool of four majorbyproducts were injected into striata of wild-type mice. It performedbetter than any compound that had been previously injected into the CNS.The products showed wide diffusion, great retention and preferentialuptake into neurons in cortex, striatum, and spinal cord, which is analmost ideal profile. Detailed characterization by HPLC and massspectrometry identified the byproducts present in the crude mixture: thedesired calciferol-hsiRNA conjugate, hsiRNA capped with a triethyleneglycol linker (TEG), and two hsiRNAs connected by a TEG linker. Thelatter compound resulted from calciferol being cleaved off duringsupport loading, leaving two active groups on which to grow hsiRNApassenger strands (FIG. 95A). After purifying each byproduct it wasdetermined that each could efficiently enter RISC in vitro (FIG. 95B),but only Di-hsiRNAs showed the wide distribution and preferentialneuronal uptake (FIG. 95C). A route to directly synthesize Di-hsiRNAwith >70% yield (FIGS. 93 and 94), confirmed by mass spectrometry, wasdevised (FIGS. 100 and 101).

A bolus ICV infusion of Di-hsiRNAs supported delivery throughout thebrain Di-branched hsiRNA (di-hsiRNA) compounds were determined tosupport wide distribution in the brain (FIGS. 68, 69 and 104A). Note thebrain injected with Cy3-Di-hsiRNA in FIG. 104A is pink throughout.Single injection of di-siRNA was detected on both ipsilateral andcontralateral to injection site indicating that spread is not limited tothe injected hemisphere but is also occurring across the midline intothe non-injected side. The lesser degree of di-siRNA accumulation on thecontralateral side, although significant, may necessitate bilateralinjections for full brain silencing. Alternative methods of injectionincluding intracerebral-ventricular may also facilitate bilateraldistribution with only one injection.

Branching was determined to be essential for enhanced brain distribution(FIG. 70). Di-hsiRNA distributed throughout the injected hemisphere ofthe mouse brain following intrastriatal injection. While a singlenon-conjugated hsiRNA can silence mRNA in primary neurons, the di-hsiRNAstructure was essential for enhanced tissue distribution and tissueretention of modified oligo nucleotides. Other conjugates such ascholesterol, although retained, showed a steep gradient of diffusionaway from the site of injection. The subtle hydrophobicity of the twosingle stranded phosphorothioated tails supported tissue retention whilealso allowing for widespread and uniform distribution throughout theipsilateral hemisphere of the injected brain.

In vivo gene silencing after single IS injections of di-hsiRNA wasstudied (FIG. 71). Single injection of di-siRNA induced robust silencingin both the striatum and cortex of mouse brain (FIGS. 72 and 73). Thislevel of efficacy has never been demonstrated previously fornon-conjugated siRNAs. Although di-hsiRNA appears visually associatedwith fiber tracts in striatum, the efficacy observed clearly indicatesthat striatal neurons internalized di-siRNA to a significant degree.

Di-hsiRNA also supported uniform spinal cord distribution (FIG. 74). Adi-hsiRNA bolus IT injection supported htt silencing in spinal cords(FIG. 75). Di-siRNA showed robust and even silencing throughout thespinal cord following intrathecal injection. A single injection ofdi-hsiRNA in the lumbar region of the spinal cord silenced mRNA to thesame degree in the cervical, thoracic and lumbar regions indicating evenand long range distribution. This accepted method of drug delivery willallow for treatment of neurodegenerative diseases affecting neurons inthe spinal cord.

Di siRNA showed a very unique cellular distribution when injectedintrastriatally into the brain (FIG. 76). Fluorescently labeled di-siRNAappeared to localize preferentially with neurons in the cortex. Thisselective feature was specific to these compounds and was not true forother siRNA conjugates, such as cholesterol, which showed no cell-typepreference.

Di-siRNA showed localization to fiber tracts in the striatum but waspresent within neuronal cell bodies in the cortex (FIG. 77). Withoutintending to be bound by scientific theory, movement to the cortex couldbe through diffusion or could be the result of retrograde transport viastriatal fiber tracts. The theory that retrograde transport is partiallyresponsible is supported by the fact that some areas of the cortexshowed full neuronal penetration while neurons in adjacent areas showedno di-hsiRNA association.

Intrathecal injection of di-hsiRNA produced similarly impressive resultsfor the spinal cord (FIG. 105A). Whereas chol-hsiRNA (the originalconjugated hsiRNA) showed a steep gradient of distribution with arelatively small amount reaching grey matter and motor neurons,di-hsiRNAs uniformly distributed throughout the spinal cord andco-localized with the motor neurons (enlarged in FIG. 105A).

The wide distribution of di-hsiRNA after a single injection wasassociated with greater than 85% silencing in the striatum, 70%silencing in the in cortex (FIG. 104B) and approximately 50% silencingin the spinal cord (FIG. 105B). While significant amounts of di-hsiRNAsaccumulated over time in the striatum, cortex, liver and kidneys (FIG.104C), no evidence of inflammation or neuronal degeneration weredetected at the highest doses tested (i.e., 400 μg ICV and 150 μg IT),which far exceed the minimum effective dose. At these levels,Chol-hsiRNAs are toxic. Based on these data, di-hsiRNAs have beenselected as a second class of chemistry for detailed characterization,optimization, and validation. A detailed characterization of di-hsiRNAswill be performed to determine the dose-response, maximum tolerated doseand therapeutic index. Cellular, molecular and biochemical assays willbe used to further measure the in vivo distribution and accumulation ofcompounds and the degree of target gene regulation.

Example 6 Evidence that Axonal Transport Contributes to Di-hsiRNADistribution in Brain

The preferential delivery of di-hsiRNAs to neurons, especially distal tothe injection site, was encouraging. In mice intrastriatally injectedwith Cy3-di-hsiRNA (FIG. 95C), we detect Di-hsiRNA in everyNeuN-positive cell (neurons) of the cortex but not in other non-neuronalcell types (e.g., glia). One interpretation of this observation is thatdi-hsiRNAs are preferentially transported along axons to distal neurons.Why would branched oligonucleotides have such a profound effect on theirdistribution? It is hypothesized that a role for cooperative binding,whereby one hsiRNA weakly binds to a receptor, and a second independentbinding event promotes internalization (Alves I D, Ciano K A,Boguslayski V, Varga E, Salamon Z, Yamamura H I, Hruby V J, Tollin G.Selectivity, cooperativity, and reciprocity in the interactions betweenthe delta-opioid receptor, its ligands, and G-proteins. The Journal ofbiological chemistry. 2004; 279:44673-82. PMID: 15317820). Cooperativebinding by covalently linked hsiRNAs might dramatically enhance the rateof cellular uptake and consequently tissue retention. This and otherhypotheses will be tested and detailed structure-activity relationshipstudies of di-hsiRNAs will be performed.

Example 7 Evidence PC-DHA and Di-hsiRNA Conjugates: Two Novel Classes ofCNS Active Oligonucleotides

As described in the data above, two novel, chemically distinct classesof therapeutic siRNAs, PC-DHA-hsiRNAs and Di-hsiRNAs, have been designedthat support wide distribution and potent gene silencing in CNS tissuesafter CSF infusion. Di-hsiRNAs appear promising but currently lack dataon safety and therapeutic index. PC-DHA-hsiRNAs have a wide therapeuticwindow (FIG. 103). This is important because antisense oligonucleotidesin clinical trials for CNS indications have a narrow therapeutic index.

To mitigate potential risk, both classes of compounds will be evaluatedin detail. The goal is to achieve greater than 70% target gene silencingat a dose of less than 200 μg/injection, greater than 10-foldtherapeutic index, and 1-month to 3-month duration of effect with abolus injection via CSF. The development of a simple technology platformthat allows straightforward and long-lasting silencing in the brain andthe spinal cord of a small animal will advance the field of neuroscienceresearch significantly. It will enable direct functional analysis of arange of novel targets with suspected involvement in brain biology andneurodegenerative disease progression. The data described hereindemonstrate that chemistry defines distribution, efficacy and safety ofoligonucleotides. Chemical variants of PC-DHA-hsiRNA and di-hsiRNA willbe evaluated to identify scaffolds with higher efficacy and widertherapeutic indices, features that are essential for future translationof this technology platform towards human therapeutics. Lastly, theperformance of several compounds will be validated in established animalmodels of neurodegenerative disease, by silencing HTT in HD.

Example 8 Characterization of PC-DHA and Di-hsiRNA Distribution,Efficacy and Safety in the Brain and the Spinal Cord

Oligonucleotide Synthesis

HsiRNA and Di-hsiRNAs will be synthesized (0.2 grams, +/−Cy3) andHPLC-purified as fully metabolically stable hsiRNAs (including 5′-E-VPas a terminal phosphate analog), followed by characterization by massspectrometry. A variety of linkers have been screened and optimalscaffolds for PC-DHA and di-hsiRNA conjugation have been identified. Thefunctionalized supports will be synthesized as shown in FIGS. 93 and 94.The following compounds will be used: HTT-10150 (HD) and PPIB-437(housekeeping control). Numbers denote the position of the human mRNAtargeted by the 5′ nucleotide of the guide strand. All compounds havebeen previously identified using optimized bioinformatics parameters(Birmingham A, Anderson E, Sullivan K, Reynolds A, Boese Q, Leake D,Karpilow J, Khvorova A. A protocol for designing siRNAs with highfunctionality and specificity. Nature protocols. 2007; 2:2068-78. PMID:17853862) and extensive experimental screening. Each siRNA targets andsilences the corresponding human, mouse and monkey mRNAs, which willsimplify future clinical development.

In addition to standard oligonucleotide synthesis systems, i.e.,Mermaid12 and Expedite, a mid-scale RNA-synthesis capability (fundedthrough an S10 grant), including an OligoPilot 100, preparative HPLCs,and high-resolution LC-MS, have been established. Large batches of novelcompounds required for the in vivo studies proposed below will besynthesized.

Optimization of Administration Route

Several routes of administration were compared and it was determinedthat a bolus infusion via CSF (ICV and IT infusion) supports the bestdegree of compound retention and distribution in CNS tissues. CSFdelivery via these routes is analogous to a “spinal tap,” a clinicallyacceptable route of administration. A side-by-side comparison of tissueretention and efficacy was compared when equivalent doses were deliveredby bolus injection or by ALZET pump over a period of one week.Significantly better tissue retention and efficacy were observed withbolus injections, consistent with data reported for ASOs (Rigo F, Chun SJ, Norris D A, Hung G, Lee S, Matson J, Fey R A, Gaus H, Hua Y, Grundy JS, Krainer A R, Henry S P, Bennett C F. Pharmacology of a centralnervous system delivered 2′-O-methoxyethyl-modified survival of motorneuron splicing oligonucleotide in mice and nonhuman primates. TheJournal of pharmacology and experimental therapeutics. 2014; 350:46-55.PMID: 24784568; PMCID: PMC4056267). Without intending to be bound byscientific theory, better performance of bolus administration over pumpadministration could be related to the mechanism of oligonucleotideuptake. For example, non-productive oligonucleotide sinks might besaturated faster by bolus than by pump infusion, thereby allowing excessoligonucleotide to be transported more readily.

To directly quantify intact guide strand in tissues, we have developedand implemented a novel and quantitative peptide nucleic acid (PNA)hybridization assay was developed and implemented (FIG. 106). The assaywas highly sensitive, with a limit of detection of less than 10 fmolehsiRNA per gram tissue. HsiRNA metabolites with full-length, partiallydegraded, 5′-phosphorylated and 5′-dephosphorylated guide strand couldbe readily quantified as separate peaks or shoulders in the HPLC trace.Using this assay the kinetics of guide strand retention in 2-mm punchbiopsies taken from regions throughout spinal cord and brain will bequantified.

Based on previous experience, accumulation of 1 to 5 μg oligonucleotideper gram tissue one week after injection is usually enough to supportproductive target silencing (FIGS. 104B, 104C). The fluorescence and PNAassays allow mapping of the distribution and quantity of conjugatedhsiRNA delivered. These studies will complement functional analyses andestablish a foundation for silencing efficacy studies.

Identify the Maximum Tolerated Dose

In pilot studies, 200 μg DHA-hsiRNA and di-hsiRNA was established as asafe dose for intrastriatal injection (data for DHA is present in FIGS.103B and 103C), 150 μg was established as a safe dose for intrathecalinjection, and 400 μg was established as a safe dose forintracerebroventricular injection. Beginning at these levels, the dosewill be increased in two-fold increments until animals show anyindications of toxicity or until drug solubility limits of approximately20 mM for PC-DHA-hsiRNA and approximately 50 mM for Di-hsiRNA arereached. Three weeks post-injection (optimal time required to seeoligonucleotide toxicity), brain tissue will be collected and the numberand viability of neurons will be assessed by staining for neuronalmarkers NeuN and DARPP-32 (Mullen R J, Buck C R, Smith A M. NeuN, aneuronal specific nuclear protein in vertebrates. Development(Cambridge, England). 1992; 116:201-11. PMID: 1483388; Weyer A,Schilling K. Developmental and cell type-specific expression of theneuronal marker NeuN in the murine cerebellum. Journal of neuroscienceresearch. 2003; 73:400-9. PMID: 12868073; Ouimet C C, Miller P E,Hemmings H C, Jr., Walaas S I, Greengard P. DARPP-32, a dopamine- andadenosine 3′:5′-monophosphate-regulated phosphoprotein enriched indopamine-innervated brain regions. III. Immunocytochemical localization.The Journal of neuroscience: the official journal of the Society forNeuroscience. 1984; 4:111-24. PMID: 6319625). Microglial activation(innate immune activation) will also be assessed by staining for IBA1(Judge A D, Bola G, Lee A C, MacLachlan I. Design of noninflammatorysynthetic siRNA mediating potent gene silencing in vivo. Moleculartherapy: the journal of the American Society of Gene Therapy. 2006;13:494-505. PMID: 16343994; Marques J T, Williams B R. Activation of themammalian immune system by siRNAs. Nature biotechnology. 2005;23:1399-405. PMID: 16273073). To test whether compounds trigger areversible, short-term inflammatory response, mice will be injected withthe maximum tolerated dose and glial activation will be examined at 6hours post-administration. Completion of this study will generate dataon the maximum tolerated dose for the two new classes of therapeutichsiRNAs described herein.

Estimate PC-DHA and Di-hsiRNAs Clearance Profiles

The residence time of RNAs in CSF and blood will be determined. Arepetitive CSF withdrawal in mice is unfeasible, therefore CSF clearancestudies will be performed using rats, adjusting the dose accordingly. 10μl of CSF will be drawn at 1, 6, 12 and 24 hours and at 1 weekpost-administration of PC-DHA- and Di-hsiRNAs using overlapping groupsof animals. Similarly, 20 μL of blood will be collected at 5 and 30 min,and at 1, 4, 12, 24, 48, 72 and 96 hours post-injection. To minimizeconcerns related to repetitive blood draws over short time periods, andto minimize the number of animals required to obtain precise data,jugular vein catheterization will be used.

Based on previous pharmacokinetic studies with related siRNA compounds,it is expected that biphasic clearance kinetics will be observed, withthe fast phase completed within four to six hours. Based on pilotstudies, it could take a month(s) for complete drug clearance. However,a one-week pilot study is enough to approximate the clearance profile.Completion of this study will generate pilot data on clearance profilesfor the two new classes of therapeutic hsiRNAs described herein.

Establishing the Dose Response

Dose-response studies will be performed to determine the optimal dosefor silencing in areas of the brain showing significant oligonucleotideaccumulation. Experiments will be performed similarly to those presentedin FIGS. 103, 104 and 105. 3-mm punch biopsies will be harvested fromthe brain and spinal column of mice injected with increasing doses ofPC-DHA and Di-hsiRNA, the levels of HTT or control mRNAs will bemeasured using the QUANTIGENE® assay.

QUANTIGENE® is a highly sensitive 96-well-based assay that uses signalamplification to detect mRNA in tissue or cell lysates directly. Aprotocol describing an automated, high-throughput (96-well) version ofthe assay that directly links TissueLyser and QUANTIGENE® was recentlypublished (Coles A H, Osborn M F, Alterman J F, Turanov A A, Godinho BM, Kennington L, Chase K, Aronin N, Khvorova A. A High-Throughput Methodfor Direct Detection of Therapeutic Oligonucleotide-Induced GeneSilencing In Vivo. Nucleic acid therapeutics. 2015. PMID: 26595721).Thus, simultaneous quantification of HTT and housekeeping mRNAs can beperformed for many tissues and/or animals. In pilot studies, it wasdetermined that eight mice per group was sufficient to detect a 40%reduction in target gene expression with 80% confidence. Id.

HTT mRNA levels will be normalized to a control housekeeping mRNA.Artificial CSF and non-targeting controls (NTC) of the same chemicalcomposition will be used to control for non-sequence-specific events.NTC hsiRNA will only be injected at the highest non-toxic concentrationto limit the number of animals used. Though NTC is a better negativecontrol, a second targeting hsiRNA (e.g., PPIB-targeting) will providesilencing data on two different targets with the same number of animals.Confirmation of silencing at the protein level is essential beforetransitioning toward animal models of disease, so Western blotting willbe performed in a similar manner as has been done for chol-hsiRNAs(Alterman J F, Hall L M, Coles A H, Hassler M R, Didiot M C, Chase K,Abraham J, Sottosanti E, Johnson E, Sapp E, Osborn Alf, Difiglia M,Aronin N, Khvorova A. Hydrophobically Modified siRNAs Silence HuntingtinmRNA in Primary Neurons and Mouse Brain. Molecular therapy Nucleicacids. 2015; 4: e266. PMID: 26623938). Completion of this study shouldidentify doses enabling functional gene silencing in different regionsof the CNS for the two new classes of therapeutic hsiRNAs describedherein.

PC-DHA and Di-hsiRNA Duration of Silencing Upon Single Administration

Most neurodegenerative disorders and disease models present a late onsetof symptoms (e.g., 3 to 9 months in mice). The duration of silencingfrom one injection and how many injections will be needed to support 6to 9 months of silencing should be determined. In general, siRNA-inducedsilencing in non-dividing cells is expected to last for month(s). Thehalf-life of loaded RISC complex is several weeks (Whitehead K A, LangerR, Anderson D G. Knocking down barriers: advances in siRNA delivery.Nature reviews Drug discovery. 2009; 8:129-38. PMID: 19180106) and lessthan 1,000 loaded RISC molecules per cell are sufficient to inducesilencing (Stalder L, Heusermann W, Sokol L, Trojer D, Wirz J, Hean J,Fritzsche A, Aeschimann F, Pfanzagl V, Basselet P, Weiler J,Hintersteiner M, Morrissey D V, Meisner-Kober N C. The roughendoplasmatic reticulum is a central nucleation site of siRNA-mediatedRNA silencing. The EMBO journal. 2013; 32:1115-27. PMID: 23511973;PMCID: 3630355; Pei Y, Hancock P J, Zhang H, Bartz R, Cherrin C,Innocent N, Pomerantz C J, Seitzer J, Koser M L, Abrams M T, Xu Y,Kuklin N A, Burke P A, Sachs A B, Sepp-Lorenzino L, Barnett S F.Quantitative evaluation of siRNA delivery in vivo. Rna. 2010;16:2553-63. PMID: 20940339; PMCID: 2995415).

Moreover, FM-hsiRNAs may provide another advantage. A cell usually takesup millions of hsiRNAs, but the vast majority are trapped in lysosomes.Conventional, partially modified hsiRNAs entrapped in lysosomes aredegraded, but FM-hsiRNAs are not. As a result, FM-hsiRNAs transientlytrapped in lysosomes form an intracellular “depot” that slowly releasesFM-hsiRNAs, making them available for RISC loading. Data from theAlnylam GalNAc trials indicate that optimized delivery to the liverprovides up to six-month efficacy from a single subcutaneous injection.The data presented herein are in line with this observation; a singleFM-hsiRNA injection provides maximal silencing for at least a month(FIG. 2D).

To measure the retention of hsiRNA and duration of silencing, three micewill be injected with the highest tolerated dose of PC-DHA- ordi-hsiRNAs, and the levels of intact guide strand in tissues will bemeasured at 1, 2 and 4 weeks and at 2, 3, 4 and 6 months using the PNAassay (FIG. 106). As soon as intact guide strand levels fall below 1 μghsiRNA per gram tissue, the study will be terminated. HTT mRNA levelswill be measured at time points where guide strand concentration isabove one μg per gram tissue in a separate study powered (n=8) toreliably detect silencing effects. Though it is expected that theduration of silencing will be at least three months, experimentalvalidation is desired.

Exploring Mechanisms of Cellular Uptake and Trafficking of Di-hsiRNAs

Di-branched hsiRNAs showed significantly enhanced retention anddistribution in CNS tissues compared to an equal dose of linker-boundsingle siRNA, indicating that cooperative binding by the covalentlylinked siRNAs or receptor dimerization drive cellular uptake (FIG. 95C).Differential uptake will be visualized and characterized using acombination of TESM microscopy (time-resolved epifluorescence structuremicroscopy) and mass spectrometry.

Develop “Antidotes” for HTT Compounds

Gene therapy approaches (i.e., permanent gene silencing) are currentlybeing considered for treatment of neurodegenerative disorders, so 1- to6-month duration of silencing seems relatively safe. Nevertheless, an“antidote” to reverse the silencing would satisfy concerns about safety.An “antidote” is also be a great tool to study gene function in vivo,allowing one to test how long a gene needs to be downregulated toproduce associated phenotypes.

Addressing similar concerns from the FDA, Alnylam has developed atechnology, called “REVERSIR®,” which enables reversal of long-termsilencing. The concept involves developing a high-affinity antisense(LNA and 2′-O-methyl/deoxy) MIXMER® fully complementary to the seedregion of the functional hsiRNA. WA panel of hsiANTIDOTEs targetingHTT10150 (and eventually other compounds) will be designed andsynthesized, and their ability to reverse silencing in vitro and in vivowill be assessed (FIG. 96). Antidotes will be synthesized in the contextof the PC-DHA conjugate to enable similar distribution properties as thePC-DHA-hsiRNA. Completion of this study will generate antidotes againstlead hsiRNA compounds to enable reversal of their in vivo activity, ifdesired.

Alternative Approach: Test Whether PC-DHA Conjugation and Di-BranchedStructure Improve Antisense-Mediated Silencing in the Brain

Antisense oligonucleotides for the treatment of neurodegenerativedisorders are in clinically advanced stages of development (Evers M M,Toonen L J, van Roon-Mom W M. Antisense oligonucleotides in therapy forneurodegenerative disorders. Advanced drug delivery reviews. 2015. PMID:25797014; Kordasiewicz H B, Stanek L M, Wancewicz E V, Mazur C, McAlonisM M, Pytel K A, Artates J W, Weiss A, Cheng S H, Shihabuddin L S, HungG, Bennett C F, Cleveland D W. Sustained therapeutic reversal ofHuntington's disease by transient repression of huntingtin synthesis.Neuron. 2012; 74:1031-44. PMID: 22726834; PMCID: PMC3383626).IONIS-HTT_(Rx) is a generation 2.5 antisense chemistry proprietary toIonis and not generally available to the academic community.

A highly potent locked-nucleic acid (LNA) GapmeR targeting HTT has beendeveloped, however. To test whether a PC-DHA conjugation and/ordi-branching can improve the distribution and retention of antisenseoligonucleotides in brain tissues and reduce their effective doses,PC-DHA- and di-LNA GapmeRs targeting HTT will be synthesized.

Completion of this example will result in the full characterization ofthe two novel oligonucleotide conjugates (i.e., PC-DHA and di-hsiRNAs)described herein in CNS (brain and spinal cord), including optimaldelivery route, drug clearance and retention, safety, dose response andduration of effect. The experimentation described herein will enable useof these chemistries for gene silencing and target validation studies inCNS in vivo, as well as provide a solid foundation toward development ofnovel therapies for HD.

Example 9 Synthesis and Characterization of a Panel of PC-DHA andDi-hsiRNA Chemical Variants to Improve Distribution and TherapeuticIndex

The data presented herein (FIGS. 92, 103, 104 and 105) indicates thatPC-DHA- and di-hsiRNA chemistry will be sufficient to reach a target of1-month to 3-month duration of effect in spinal cord, striatum, andcortex, which is sufficient for functional genomics studies in vivo.This alone is a significant achievement, but future translation of thistechnology platform toward human therapeutics represents another levelof complexity. Before we translate the technology, we will optimize thechemistry for (i) the widest possible therapeutic index and (ii)enhanced distribution to support delivery to large brains.

Slight changes in the chemical scaffold of a conjugate can profoundlyaffect tissue distribution as was demonstrated by functionalizing DHAwith phosphatidylcholine (FIG. 92). Capitalizing on these syntheticplatforms, a panel of PC-DHA- and di-hsiRNA variants will be synthesizedto further optimize therapeutic index and wide tissue distribution.

PC-DHA Optimization

There are two essential components to the PC-DHA structure:phosphatidylcholine and DHA (see structure in FIG. 92). The synthesisapproach described herein (FIGS. 93 and 94) will allow these chemistriesto be varied independently. Little to no information exists in theliterature on the structure-function relationship of oligonucleotideconjugates, but a large body of information exists describing howpolymer structures and lipid compounds affect lipid-particleformation⁴⁸. The studies indicate that the length of lipid has a majorimpact on overall formulation efficacy.

Polyunsaturated bonds are essential for enhanced hsiRNA distribution inCNS tissues. Conjugation of DCA, a fully saturated analog of DHA, doesnot promote wide distribution in CNS. Conjugation of EPA, two carbonsshorter than DHA, leads to an interesting distribution profile, butefficacy has not yet been tested. A panel ofphosphatidylcholine-modified polyunsaturated fatty acid variants,changing the length of the lipid tail from 10 to 22 carbons and thenumber of polyunsaturated bonds from 0 to 4 will be synthesized. Theprecursors for these synthesis reactions are all commercially available.These compounds will reveal how the length of the lipid tail length andnumber of polyunsaturated bonds affects oligonucleotide distribution inthe CNS.

Second, a systematic substitution of the choline entity will beperformed for a range of modifications, mostly favoring naturallyoccurring chemical scaffolds, e.g., phosphatidylserine,phosphatidylinositol and phosphatidyl amine. Most of these syntheses canbe performed in parallel, creating a library of compounds with fixedlipid tail composition and a variety of head groups. This library willbe used to define the importance of head groups on the in vivoperformance of hsiRNA conjugates.

It is well known that, regardless of the nature of the chemistry orformulation for delivery, the vast majority of internalizedoligonucleotides are not biologically available. Endosomolytic,peptide-modified polymers have been used by Arrowhead ResearchCorporation (Wong S C, Klein J J, Hamilton H L, Chu Q, Frey C L,Trubetskoy V S, Hegge J, Wakefield D, Rozema D B, Lewis D L.Co-injection of a targeted, reversibly masked endosomolytic polymerdramatically improves the efficacy of cholesterol-conjugated smallinterfering RNAs in vivo. Nucleic acid therapeutics. 2012; 22:380-90.PMID: 23181701) to enhance systemic efficacy of co-administeredcholesterol-modified siRNA compounds. Building on this concept, alibrary of linkers varying the number and composition of endosomolyticpeptides was synthesized. Most variants had no impact on chol-hsiRNAefficacy, but the best lead (FIG. 97A) enhanced silencing upon passiveuptake greater than 10-fold (FIG. 97B). Without intending to be bound byscientific theory, the enhanced activity likely results from theincreased bioavailability of internalized chol-hsiRNA, as the modifiedlinker did not increase the efficacy by lipid-mediated uptake (FIG. 97B)or the overall amount of oligonucleotide internalized. This linker willbe combined with the most optimal combination of lipid length and headgroup.

Di-hsiRNA Optimization

The two hsiRNAs in the di-hsiRNA compound are connected asymmetrically:one by a phosphate bond and the other by a phosphoramidate bond (FIGS.93, 94 and 103A). To establish whether the phosphoramidate bond isnecessary, a di-branched compound in which the hsiRNAs are bothconnected to the linker via phosphates is being currently tested(modified synthesis scheme based on that in FIGS. 93 and 94). Showingthat the phosphoramidate bond is not essential would simplifystructure-activity relationship studies, as a large number ofcommercially available precursors can be used. Nevertheless, arequirement for the phosphoamide bond would be interesting becausephosphoamide is much less stable in acidic conditions and is expected topromote release of compounds from endosomes.

A panel of variants will be synthesized, testing the followingparameters: number of hsiRNAs (2, 3, 4 or 6, using two- and three-branchdividers available from Glen Research); and the chemical nature of thelinker connecting the oligonucleotides (TEG, saturated and non-saturatedalkyl chain, charged, non-charged, lengths from 3 to 30 carbons andproton sponges). The minimal number of phosphorothioate bonds requiredfor uptake will also be identified. It was already determined thatphosphorothioate bonds were essential for passive uptake and efficacy ofhsiRNA, so it is suspected that cooperative binding of twophosphorothioate tails drives the enhanced distribution and uptake ofDi-hsiRNAs. However, phosphorothioate bonds also drive the toxicity ofantisense oligonucleotides (Geary R S, Norris D, Yu R, Bennett C F.Pharmacokinetics, biodistribution and cell uptake of antisenseoligonucleotides. Advanced drug delivery reviews. 2015; 87:46-51). Thus,optimizing and reducing the number of phosphorothioates is a reasonablepath toward enhancing the therapeutic index.

Evaluation of Efficacy of PC-DHA- and Di-hsiRNA Variants

Tissue culture experiments will be used to confirm safety and efficiententry into the RISC complex. Each compound will then be injected ICV atthe minimum effective and maximum tolerated doses established asdescribed herein. Compounds that are efficacious at lower concentrationsor/and nontoxic at higher concentrations will be selected for detailedstudies. Lastly, the ability of the most promising compounds todistribute through a large brain (e.g., sheep) will be assessed. A sheepmodel has been designed to evaluate the distribution of AAV-htt vectors.The PNA assay described herein will be used to measure the levels ofcompound in biopsies from different regions of the sheep brain after abolus ICV infusion.

Completion of this example is expected to: (i) inform on chemicalstructures that define in vivo efficacy of PC-DHA- and Di-hsiRNAs; and(ii) generate versions of these compounds with enhanced distribution,efficacy and therapeutic index.

Example 10 Development of Candidate Pre-Clinical Compounds forHuntington's Disease Models

Evaluation of Novel Conjugate of hsiRNA HTT10150 in Huntington's DiseaseAnimal Models

The hyper-functional, FM-hsiRNA for Htt, HTT-10150, is described herein.Multiple HD animal models currently running in his lab (Chang R, Liu X,Li S, Li X J. Transgenic animal models for study of the pathogenesis ofHuntington's disease and therapy. Drug design, development and therapy.2015; 9:2179-88. PMID: 25931812; PMCID: PMC4404937), including YAC128(Hodgson J G, Agopyan N, Gutekunst C A, Leavitt B R, LePiane F,Singaraja R, Smith D J, Bissada N, McCutcheon K, Nasir J, Jamot L, Li XJ, Stevens M E, Rosemond E, Roder J C, Phillips A G, Rubin E M, Hersch SM, Hayden M R. A YAC mouse model for Huntington's disease withfull-length mutant huntingtin, cytoplasmic toxicity, and selectivestriatal neurodegeneration. Neuron. 1999; 23:181-92. PMID: 10402204),BACHD (Hult S, Soylu R, Bjorklund T, Belgardt B F, Mauer J, Bruning J C,Kirik D, Petersen A. Mutant huntingtin causes metabolic imbalance bydisruption of hypothalamic neurocircuits. Cell metabolism. 2011;13:428-39. PMID: 21459327; Hult Lundh S, Nilsson N, Soylu R, Kirik D,Petersen A. Hypothalamic expression of mutant huntingtin contributes tothe development of depressive-like behavior in the BAC transgenic mousemodel of Huntington's disease. Human molecular genetics. 2013;22:3485-97. PMID: 23697793; Gray M, Shirasaki D I, Cepeda C, Andre V M,Wilburn B, Lu X H, Tao J, Yamazaki I, Li S H, Sun Y E, Li X J, Levine MS, Yang W. Full-length human mutant huntingtin with a stablepolyglutamine repeat can elicit progressive and selectiveneuropathogenesis in BACHD mice. The Journal of neuroscience: theofficial journal of the Society for Neuroscience. 2008; 28:6182-95.PMID: 18550760; PMCID: PMC2630800), and recently established allelicseries including Q140 (Website: chdifoundation.org), will be used.

Based on optimal parameters identified as described herein, a bolus ICVinjection of HTT-10150 will be administered into each Huntington's mousemodel and the mice will be assayed for Htt silencing, Huntington'sbehavior and/or onset of Huntington's-associated phenotypes, andvalidated histological parameters. A set of validated assays have beendesigned to detect differential expression of YAC128 and Q140 mutantmRNAs and wild-type Htt mRNA. A panel of behavioral assays has beendesigned to assess motor function, including rotarod, elevated platform,and open field assays (Sah D W, Aronin N. Oligonucleotide therapeuticapproaches for Huntington disease. The Journal of clinicalinvestigation. 2011; 121:500-7. PMID: 21285523; PMCID: 3026739;Kordasiewicz H B, Stanek L M, Wancewicz E V, Mazur C, McAlonis M M,Pytel K A, Artates J W, Weiss A, Cheng S H, Shihabuddin L S, Hung G,Bennett C F, Cleveland D W. Sustained therapeutic reversal ofHuntington's disease by transient repression of huntingtin synthesis.Neuron. 2012; 74:1031-44. PMID: 22726834; PMCID: PMC3383626). One groupof mice will be treated at age three months to assess diseaseprevention, and another group of mice will be treated at age six monthsto assess disease reversal. HTT aggregates will be assessed byimmunohistochemical staining using a commercially availableanti-polyglutamine antibody (3B5H10). HTT-10150 hsiRNA conjugates willbe re-administered, if necessary. Control groups will include miceinjected with PBS and non-targeting control compound having identicalchemistry as the HTT-10150 hsiRNA conjugate. The best lead will bere-test them independently by another group in several behavioral modelsof Huntington's disease.

Completion of this example, together with efficacy, safety andduration-of-effect studies, will generate a set of data sufficient tomove the optimized hsiRNA HTT-10150 into pre-clinical development.Currently, the best available program for oligonucleotide-basedtreatment of Huntington's disease is the 2′-O-methoxyethyl Gapme® (Id.),which will be used as a benchmark. The IONIS-HTT_(Rx) compound hasrecently initiated Phase 1 clinical trials in which patients receive abolus spinal tap injection and a reduction in HTT levels in CSF servesas a biomarker for proof of concept. This establishes a clinical pathforward in the development of oligonucleotide therapeutics to treatHuntington's disease. Initially, it is planned to silence both mutantand wild-type Htt, similar to the IONIS approach. The scaffold describedherein will also be used to generate compounds that selectively silencemutant HTT by SNP discrimination. Indeed, five SNP alleles are linked totoxic CAG expansion in 75% of Huntington's disease patient mutations(Pfister E L, Kennington L, Straubhaar J, Wagh S, Liu W, DiFiglia M,Landwehrmeyer B, Vonsattel J P, Zamore P D, Aronin N. Five siRNAstargeting three SNPs may provide therapy for three-quarters ofHuntington's disease patients. Current biology: CB. 2009; 19:774-8.PMID: 19361997; PMCID: PMC2746439).

INCORPORATION BY REFERENCE

The contents of all cited references (including literature references,patents, patent applications, and websites) that maybe cited throughoutthis application are hereby expressly incorporated by reference in theirentirety for any purpose, as are the references cited therein. Thedisclosure will employ, unless otherwise indicated, conventionaltechniques of immunology, molecular biology and cell biology, which arewell known in the art.

The present disclosure also incorporates by reference in their entiretytechniques well known in the field of molecular biology and drugdelivery. These techniques include, but are not limited to, techniquesdescribed in the following publications:

-   Atwell et al. J. Mol. Biol. 1997, 270: 26-35;-   Ausubel et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John    Wiley &Sons, NY (1993);-   Ausubel, F. M. et al. eds., SHORT PROTOCOLS IN MOLECULAR BIOLOGY    (4th Ed. 1999) John Wiley & Sons, NY. (ISBN 0-471-32938-X);-   CONTROLLED DRUG BIOAVAILABILITY, DRUG PRODUCT DESIGN AND    PERFORMANCE, Smolen and Ball (eds.), Wiley, New York (1984);-   Giege, R. and Ducruix, A. Barrett, CRYSTALLIZATION OF NUCLEIC ACIDS    AND PROTEINS, a Practical Approach, 2nd ea., pp. 20 1-16, Oxford    University Press, New York, N.Y., (1999);-   Goodson, in MEDICAL APPLICATIONS OF CONTROLLED RELEASE, vol. 2, pp.    115-138 (1984);-   Hammerling, et al., in: MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS    563-681 (Elsevier, N.Y., 1981;-   Harlow et al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor    Laboratory Press, 2nd ed. 1988);-   Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST    (National Institutes of Health, Bethesda, Md. (1987) and (1991);-   Kabat, E. A., et al. (1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL    INTEREST, Fifth Edition, U.S. Department of Health and Human    Services, NIH Publication No. 91-3242;-   Kontermann and Dubel eds., ANTIBODY ENGINEERING (2001)    Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).-   Kriegler, Gene Transfer and Expression, A Laboratory Manual,    Stockton Press, NY (1990);-   Lu and Weiner eds., CLONING AND EXPRESSION VECTORS FOR GENE FUNCTION    ANALYSIS (2001) BioTechniques Press. Westborough, Mass. 298 pp.    (ISBN 1-881299-21-X).-   MEDICAL APPLICATIONS OF CONTROLLED RELEASE, Langer and Wise (eds.),    CRC Pres., Boca Raton, Fla. (1974);-   Old, R. W. & S. B. Primrose, PRINCIPLES OF GENE MANIPULATION: AN    INTRODUCTION TO GENETIC ENGINEERING (3d Ed. 1985) Blackwell    Scientific Publications, Boston. Studies in Microbiology; V.2:409    pp. (ISBN 0-632-01318-4).-   Sambrook, J. et al. eds., MOLECULAR CLONING: A LABORATORY MANUAL (2d    Ed. 1989) Cold Spring Harbor Laboratory Press, NY. Vols. 1-3. (ISBN    0-87969-309-6).-   SUSTAINED AND CONTROLLED RELEASE DRUG DELIVERY SYSTEMS, J. R.    Robinson, ed., Marcel Dekker, Inc., New York, 1978-   Winnacker, E. L. FROM GENES TO CLONES: INTRODUCTION TO GENE    TECHNOLOGY (1987) VCH Publishers, NY (translated by Horst    Ibelgaufts). 634 pp. (ISBN 0-89573-614-4).

Equivalents

The disclosure may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting of the disclosure. Scope of the disclosure is thusindicated by the appended claims rather than by the foregoingdescription, and all changes that come within the meaning and range ofequivalency of the claims are therefore intended to be embraced herein.

1-76. (canceled)
 77. A recombinant adeno-associated virus (rAAV) vectorcomprising a stem-loop structure comprising a nucleotide sequenceencoding an RNA duplex comprising a sense strand and an antisensestrand; wherein the antisense strand is between 16 and 22 nucleotides inlength and comprises a region of complementarity; and wherein the regionof complementarity in the antisense strand is complementary to at least16 contiguous nucleotides of 5′ GCCUGCUAGCUCCAUGCUUA 3′ (SEQ ID NO: 17).78. The rAAV vector of claim 77, wherein the region of complementarityin the antisense strand is complementary to at least 17 contiguousnucleotides of SEQ ID NO:
 17. 79. The rAAV vector of claim 77, whereinthe region of complementarity in the antisense strand is complementaryto at least 18 contiguous nucleotides of SEQ ID NO:
 17. 80. The rAAVvector of claim 77, wherein the sense strand is between 16 and 22nucleotides in length and comprises a nucleotide sequence which is atleast 80% identical to SEQ ID NO:
 17. 81. The rAAV vector of claim 77,wherein the sense strand is between 17 and 22 nucleotides in length andcomprises a nucleotide sequence which is at least 85% identical to SEQID NO:
 17. 82. The rAAV vector of claim 77, wherein the sense strand isbetween 18 and 22 nucleotides in length and comprises a nucleotidesequence which is at least 90% identical to SEQ ID NO:
 17. 83. The rAAVvector of claim 77, wherein the antisense strand comprises a nucleotidesequence which is at least 85% identical to 5′ UAAGCAUGGAGCUAGCAGGC 3′(SEQ ID NO: 328).
 84. The rAAV vector of claim 77, wherein the antisensestrand comprises a nucleotide sequence which is at least 90% identicalto 5′ UAAGCAUGGAGCUAGCAGGC 3′ (SEQ ID NO: 328).
 85. The rAAV vector ofclaim 77, wherein the antisense strand comprises a nucleotide sequencewhich is at least 85% identical to 5′ UAAGCAUGGAGCUAGCAGGC 3′ (SEQ IDNO: 328); and wherein the sense strand is between 17 and 22 nucleotidesin length and comprises a nucleotide sequence which is at least 85%identical to SEQ ID NO:
 17. 86. The rAAV vector of claim 77, wherein theantisense strand comprises a nucleotide sequence which is at least 90%identical to 5′ UAAGCAUGGAGCUAGCAGGC 3′ (SEQ ID NO: 328); and whereinthe sense strand is between 18 and 22 nucleotides in length andcomprises a nucleotide sequence which is at least 90% identical to SEQID NO:
 17. 87. The rAAV vector of claim 77, wherein the sense strand,the antisense strand, or both the sense strand and the antisense strand,are each 21 nucleotides in length.
 88. The rAAV vector of claim 77,wherein the sense strand and the antisense strand comprise at least onemismatched base pair.
 89. A recombinant adeno-associated virus (rAAV)comprising the rAAV vector of claim 77 and an AAV capsid.
 90. Apharmaceutical composition comprising the rAAV of claim 89 and apharmaceutically acceptable carrier.
 91. A method for inhibitingexpression of HTT gene in a cell, the method comprising introducing intothe cell the rAAV vector of claim
 77. 92. A method of treatingHuntington's Disease in a subject, the method comprising administeringto the subject a therapeutically effective amount of the rAAV of claim89.
 93. The method of claim 92, wherein the rAAV is administeredintravenously.
 94. The method of claim 92, wherein administering therAAV to the subject causes a decrease in HTT gene mRNA in the striatum,the cortex, or both the striatum and the cortex of the subject.
 95. Themethod of claim 91, wherein the cell is: (i) a CNS cell; (ii) a neuronalcell or an astrocyte; or (iii) in a subject.
 96. The method of claim 95,wherein the subject has Huntington's Disease.